Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes Postprint (Author accepted manuscript)

Ranjha, Lepakshi; Institute for Research in Biomedicine (IRB), Faculty of Biomedical Sciences, Università della Svizzera italiana, Switzerland

Howard, Sean Michael; Institute for Research in Biomedicine (IRB), Faculty of Biomedical Sciences, Università della Svizzera italiana, Switzerland

Cejka, Petr; Institute for Research in Biomedicine (IRB), Faculty of Biomedical Sciences, Università della Svizzera italiana, Switzerland - Institute of Biochemistry, Department of Biology, ETH Zürich, Switzerland

Originally published in:

Chromosoma

2018 / Vol. 127 / n. 2 (June) / pp. 187–214

Published version: https://doi.org/10.1007/s00412-017-0658-1

Online publication: 2018-01-11

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Manuscripttitle:

Main steps in DNA double-strand break repair: an introduction to homologous

recombinationandrelatedprocesses

Authors:

LepakshiRanjha1*,SeanHoward1*,PetrCejka1,2

Affiliation:

1Institute for Research in Biomedicine Università della Svizzera Italiana, Bellinzona,

Switzerland

2DepartmentofBiology,InstituteofBiochemistryETHZurich,Zurich,Switzerland

Correspondence:

PetrCejka,petr.cejka@irb.usi.ch,phone:+41918200342,fax:+41918200305

*Equalcontribution

Keywords:Homologousrecombination,end-joining,DNAdouble-strandbreakrepair,

meiosis,replicationstress,DNAendresection,DNAstrandexchange

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Abstract

DNA double-strand breaks arise accidentally upon exposure of DNA to radiation,

chemicals or result from faulty DNA metabolic processes. DNA breaks can also be

introduced in a programmedmanner, such as during thematuration of the immune

system,meiosis or cancer chemo- or radiotherapy. Cells have developed a variety of

repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly,

vegetativecellsemploymechanismsthatrestorethe integrityofbrokenDNAwith the

highest efficiency at the lowest cost of mutagenesis. In contrast, meiotic cells or

developing lymphocytes exploitDNAbreakage to generatediversity.Here,we review

the main pathways of eukaryotic DNA double-strand break repair with the focus on

homologousrecombinationanditsvarioussub-pathways.Wehighlightthedifferences

between homologous recombination and end-joining mechanisms including non-

homologous end-joining andmicrohomology-mediated end-joining, and offer insights

intohowthesepathwaysareregulated.Finally,weintroducenon-canonicalfunctionsof

therecombinationproteins,inparticularduringDNAreplicationstress.

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1OverviewofDNAdouble-strandbreakrepairpathways

1.1FormationandtypesofDNAbreaksOurDNA is under a constant threat of damage from radiation, chemicals or aberrant

DNAmetabolicprocesses(JacksonandBartek2009;CicciaandElledge2010;Tubbsand

Nussenzweig2017).AlargefractionofDNAlesionsinvolvemodificationsofDNAbases,

suchasoxidation,ultravioletlight-inducedpyrimidinedimers,methylationorcreation

of abasic sites, which leave the phosphodiester backbone intact. Other abnormalities

resultinthedisruptionofthephosphodiesterbackbone.Themostcommonoftheseare

DNA single-strandbreaks(SSBs),whereonlyoneDNA strand is interrupted (Fig.1a).

SSBs normally do not compromise the integrity of double-stranded DNA (dsDNA).

However, ifanSSBisleftunrepairedandthelesionisencounteredbyDNAmachinery

that separates the DNA duplex into two component single-strands (ssDNA), such as

duringDNAreplication,anSSBcanbeconvertedintoaone-endedDNAdouble-strand

break(DSB),seeFig.1b.BothSSBsandDSBscanariseasaresultofionizingradiation

(IR), which may occur either directly or indirectly via generation of reactive oxygen

species (Ward 1988). As a result, radiation-induced DNA damage results in complex

lesions,wherebothSSBsandDSBsareaccompaniedbyoxidativeDNAdamage(Oliveet

al. 1990; Olive 1998). The most common source of accidental IR exposure is the

radioactive radon gas that accumulates in certain locations in the basements of old

homes (Jackson and Bartek 2009). IR remains one of the most effective treatments

during anti-cancer therapy, as it preferentially affects rapidly dividing cancer cells

(JacksonandBartek2009;Baskaretal.2014).SSBsandDSBsalsoariseduringaberrant

DNA topoisomerase reactions, which can occur spontaneously or upon exposure to

specific inhibitors that are often used as anti-cancer chemotherapeutics (Holm et al.

1989;Canela et al. 2017). Finally,DNAbreaksunder certain conditions result froma

nucleaseattackingdiverseDNA intermediates, includingstalledDNAreplication forks.

Aswillbe introduced inchapter7,DSB-likestructuremayalsoariseduringaprocess

termedreplicationforkreversalwithouttemplateDNAbreakage.

Dependingonthemechanismofbreakformation,aDSBcaneitherbeone-endedor

two-ended(Fig.1b,c).Mostone-endedDSBsarisewhenDNAreplicationencountersan

SSBandthereplicationforkfallsapart,orwhenaDNAreplicationforkstallsandoneof

thearmsiscleavedbyanuclease.Two-endedDSBstypicallyformwhenbothstrandsof

linear dsDNA are broken simultaneously, or when two ssDNA breaks form in an

immediate proximity; in this case, the broken end would contain a short stretch of

overhangingssDNA.Additionally, dependingon themechanismof theDSB formation,

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DNAendsmaybeeitherchemically"clean"or"dirty".Theso-calledcleanDNAbreaks,

apart from the broken phosphodiester backbone, bear normal DNA chemistry. Dirty

DNA ends instead contain additional adducts that may include anything from small

chemicalgroupstocovalentlyattachedproteins.DNAbreaksinducedbyDNAnucleases

aregenerallyclean,butmanyDSBsinducedbyionizingradiationaredirty(Oliveetal.

1990). Likewise, DSBs induced by aberrant DNA topoisomerase reactions result in a

covalently attachedDNA topoisomeraseat thebreak end(Tse et al. 1980).Aswill be

describedbelow,themechanismofDSBrepairdependslargelyonwhetherthebreakis

one-ortwo-ended,chemicallycleanordirty,aswellasthecellcyclestage.

OurcellshavedevelopednumerousmechanismsforrepairingDSBswithaminimal

loss of genetic information. However, if a DSB is repaired incorrectly, it can lead to

mutationsandchromosomalrearrangements,resultinginaberrantregulationofcellular

growth and cancer development or even cell death (Jackson and Bartek 2009).

Therefore, accurate recognition and repair of DSBs is essential to maintain genomic

integrityandpreventtumorigenesis.

1.2End-joiningandhomologousrecombinationmechanismsrepairDNAdouble-

strandbreaks

Eukaryotic cells use twomainprocesses for DSB repair, end-joining and homologous

recombination(HR),seeFig.2a.Theend-joiningpathwayscanbe furtherdivided into

canonical non-homologous end-joining (NHEJ) and alternative non-homologous end-

joining(altNHEJ),alsotermedmicrohomology-mediatedend-joining(MMEJ).TheMMEJ

abbreviation will be used hereafter in this review. As the name indicates, NHEJ and

MMEJ involve thedirectligationoftwoDSBendswithlittleornosequencehomology

required, see below for details (Chang et al. 2017). Therefore, a key feature of end-

joiningisthatarepairtemplate,suchasthesisterchromatid, isnotrequired,soitcan

occurduringanyphaseofthecellcycle.BothNHEJandMMEJprocessestypicallyleadto

a limited loss of genetic information resulting in short deletions at the DSB site.

Additionally, becauseNHEJandMMEJmechanismsare template-independent, ligation

of the incorrect ends, if multiple DSBs are present, can generate large deletions or

chromosomalrearrangements(Changetal.2017).The end-joiningpathwayscanonly

repair two-ended DSBs, and abnormal structures at the break sites ("dirty" ends,

especially protein blocks) may inhibit this type of repair. In summary, end-joining

pathwaysrepresentfast,butpotentiallymutagenicDSBrepairprocesses(Fig.2a).

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In contrast, HR requires a homologous sequence as a template for repair

(Kowalczykowski 2015). This allows the recombination machinery to restore any

missinggeneticinformationinthevicinityofthebreaksite,and,asaresult,HRislargely

accurate.Inmostcasesinvegetativelygrowingcells,thesisterchromatidisusedasthe

repair template. This restricts recombination to cell cycle stages when the sister

chromatidisavailable,whichincludestheSandG2phases,andthusnecessitatesastrict

controlmechanism(Orthweinetal.2015).HRiscapableofrepairingbothone-andtwo-

endedDSBs and canalso repairdirtyDNAbreaks; inparticular thosewith covalently

attachedproteins. In contrast to end-joining,HR ismechanisticallymore complicated,

involves a larger number of enzymes, and is thus comparatively slower but more

accurate(Kowalczykowski2015;Changetal.2017).

Recent yearsbroughtbreakthroughs in genomeediting technologies,whichwere

spearheadedbythedevelopmentofengineerednucleasessuchaszincfingernucleases

(ZFNs) or transcription-activator like effector nucleases (TALENs) (Lombardo et al.

2007;Bedelletal.2012).Themajorityofgenomeeditingapplicationsnowexploitthe

bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9

system (Jinek et al. 2012; Mali et al. 2013). The common denominator of these

approaches is the capacity to induce a site-specificDSB.The choiceof theDSB repair

pathwaythendictatestheresultofediting(Fig.2b).ImpreciserepairbyNHEJorMMEJ

givesriseto"indel"mutations(insertionordeletions,althoughdeletionsaremuchmore

common)at thebreaksite,whichmaydisrupt thereading frameof the targetedgene

and thus result in a loss of function. Conversely, if a DNA template is provided, the

recombinationmachinerymaygetinvolved,whichcanmediateprecisealterationofthe

DNA sequence, including introductionofDNA segmentsor correctionof apathogenic

mutation(Fig.2b).Theadvanceofthesegenomeeditingtechnologiesbroughtrenewed

interest in understanding the balance between the DSB repair pathways, as the

inhibitionofMMEJandNHEJrepairpromotesHR-basedprecisegenomeediting(Chuet

al.2015;Mateos-Gomezetal.2017;Schimmeletal.2017;Zelenskyetal.2017).

Thekeyprocess thatstandsat thecrossroadsbetweenend-joiningandHR is the

initialprocessingoftheDNAbreak(Cejka2015).NHEJandMMEJrequirelittleDNAend

processing (see chapter 2 for more details). In contrast HR (including all its sub-

pathways) is initiated by DNA resection at the break site that exposes long tracts of

ssDNA.ThisssDNAisthenusedinasearchforahomologousdsDNAsequence(suchas

thesisterchromatid)thatservesasatemplateforthelargelyaccuraterepairoftheDSB

by the recombinationpathway.At the same time, extendedDNAend resectionmakes

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the DNA break generally non-ligatable and inhibits end-joining. Therefore, extensive

DNAendresectioncommitsDSBrepair to theHR-mediatedpathwayandaccordingly,

theinitiationofDNAendresectionisstrictlycontrolled(SymingtonandGautier2011;

Chapmanetal.2012;Shibata2017).ThiscontrolmechanismallowsHRtobeinitiated

onlywhenarepairtemplateisavailable(S/G2phase),andthuslimitsthepotentialfor

illegitimate recombination (i.e., recombination between not fully homologous DNA

sequences).Thisiselegantlyachievedthroughtheactivationofkeyresectionfactorsby

cyclin-dependentkinase(CDK)-catalyzedphosphorylation(Iraetal.2004;Huertasetal.

2008).Itshouldbepointedoutthatthissimplemodelhasbeenchallenged,andthereis

evidencethatlimitedorevenextendedDNAendresection,occurringintheG1phase,is

involvedinthecanonicalNHEJpathway(Biehsetal.2017).Elucidatingthedetailsand

theregulationoftheseprocesseswillbeanexcitingdirectionoffutureresearch.Since

misregulation of these DSB repair pathways is believed to result in genome

rearrangementsthataretypicalinmanycancertypes,understandingtheseprocessesis

highlyrelevantforhumanhealth.

1.3End-joiningandrecombinationprocessesinvolveseveralsubpathways

BothHRandend-joiningprocessesarenotsimplelinearpathways.Bothcanbedivided

intoseveralsub-pathways,whichsignificantlydifferintermsofrepairmechanismsand

enzyme requirements. Here, we will introduce the basic principles of these repair

processes;amoredetaileddescriptionthat includes thekeyenzymaticplayerswillbe

providedinsubsequentchapters.

With regard to the end-joining mechanisms, canonical NHEJ significantly differs

fromMMEJ(Fig.3).WhereascanonicalNHEJrequiresnoorverylimitedhomology(less

than4nt)betweenthebrokenDNAmolecules,MMEJwasfoundasaDNAend-joining

event that occurs independently of the key NHEJ factors and usually involves short

stretches ofmicrohomology (2-20 nt) between the two brokenDNA ends tomediate

repair(Seoletal.2017).Therefore,MMEJissometimesconsideredaseparateprocess

thatstandsbetweentheNHEJandHRpathways.

Recombination processes, in a broad sense, can be divided into single-strand

annealing (SSA), synthesis-dependent strand annealing (SDSA), break induced

replication (BIR) and canonical HR (also called canonical DNA double-strand break

repair, DSBR) (Kowalczykowski 2015). Themain conceptual differences between the

mechanismsofthesesub-pathwaysareschematicallyillustratedinFig.4.Dependingon

whether the flanking sequences of the recombiningDNAmolecules are exchanged or

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not, recombination leads to crossover or non-crossover recombination products. A

crossover is defined as an eventwhere thedistalarmof thebrokenDNA is swapped

withthedistalarmofthetemplateDNAmolecule.AsschematicallydepictedinFig.4d,a

crossover results in the "blue" DNA molecule ultimately joined with the "red" one.

Crossovers that occur between two homologous loci of sister chromatids give rise to

"equal"sisterchromatidexchanges,whicharemutagenicallysilent(Fig.4).Incontrast,

crossovers that occur between two ectopic loci (non-homologous loci, such as in

repetitive sequences) of sister chromatids give rise to "unequal" sister chromatid

exchanges. Likewise, crossovers resulting from recombination between non-sister

(homologous) chromosomesalso lead to grossgenome rearrangements. Furthermore,

events when genetic information from one DNA molecule gets unidirectionally

transferredintoanotherDNAmoleculearereferredtoasgeneconversions.Thisoccurs

when a DNA sequence is copied from a donor template to the brokenDNAmolecule

duringBIR,SDSAorcanonicalHR.Asabove,geneconversioncanbemutagenicwhenan

ectopic site of a sister chromatid or non-sister chromosome is used as a template.

MutationsarisinginthecourseofDSBrepairbyeitherNHEJorHRmaygiverise toa

lossof heterozygosity,which is a keydriverof tumorigenesis (FearonandVogelstein

1990).

SSA involves DNA end resection to reveal repetitive DNA sequences, which are

subsequentlyannealed.TheresultingDNAflapsarecleavedandthestrandsareligated.

SSAleadsto thedeletionof theDNAsequencebetweenthe tworepeats,and is thusa

verymutagenicrepairprocess(Fig.4a).SSAisrestrictedtosituationswhentworepeats

flankthebreaksite,andcansuccessfullyrestoretheintegrityofDSBswithinrepetitive

DNA sequences. Although SSA is conceptually similar to MMEJ, it is usually grouped

togetherwithrecombinationmechanismsbecauseofenzymerequirements(seebelow).

Additionally,unlikeMMEJ,SSArequiresextensiveDNAendresection,which isshared

amongallrecombinationsub-pathways(Bhargavaetal.2016).

Inmostcases,DNAendresectionisfollowedbythe invasionof theresectedDNA

into the dsDNA template, forming a joint molecule intermediate (Kowalczykowski

2015).This initiatesDNAsynthesis,whichrestoresmissinggenetic informationat the

breaksite.InSDSA,thejointmoleculeintermediateisdestabilizedandthenascentDNA

isannealed to theotherendof thebrokenDNAmolecule(Fig.4b).SDSA isoneof the

least mutagenic recombination sub-pathways, resulting in a non-crossover repair

product. InBIR,DNA synthesisproceeds all theway to the endof the templateDNA,

copying the sequence of the entire chromosome arm (Fig. 4c). BIR is thus a unique

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pathway for repair of one-endedDSBs resulting fromcollapsedDNA replication forks

(Sakofsky and Malkova 2017). Additionally, BIR allows telomere lengthening in the

absenceoftelomerase(SakofskyandMalkova2017).ThegeneticresultofBIRisanon-

reciprocalcrossover.

In the canonical recombinational DSBR pathway, the joint molecule (D-loop)

intermediate is processed into double Holliday junctions (dHJs), which may be

processedintonon-crossoversorcrossovers,dependingonthedHJprocessingpathway

utilized (see chapter 5) (Fig. 4d) (Szostak et al. 1983). Although the various repair

events described above share some processing steps, they have different degrees of

mutagenicpotential.Todate,manyofthekeyrepairfactorsforeachpathwayhavebeen

identified;however,howacelldetermineswhichpathwaytouseforDSBrepairisstill

poorlyunderstood.TheinitialprocessingofbrokenDNAendsseemstobethekeystep

thatdetermineswhichpathwayisusedtorepairaDSB,andwillbedescribedinthenext

section.

2ProcessingofDNAbreaksforrepair

DNAendresectioninvolvesthedegradationofthe5'-terminatedDNAstrandinthe5'to

3'directionfromthebreaksitetogeneratea3'ssDNAoverhang.Generationofthis3'-

terminatedssDNAisessentialtoallowfortheusageofhomologousDNAsequencesfor

repair.ThehomologymaybeeitherbetweenthetworesectedendsofthebrokenDNA

molecule,suchasincaseofMMEJorSSA,orbetweentheresectedbrokenDNAmolecule

and an intact dsDNA template, such as in case of BIR, SDSA and canonical

recombinational DSBR. Nucleolytic resection of DNA ends typically inhibits canonical

NHEJ.

2.1ProcessingofDNAendsforcanonicalNHEJ

ManycanonicalNHEJeventsinvolvelittleornoprocessingofthebrokenDNAends.The

initialstepofNHEJinvolvesthebindingoftheDNAendsbytheKu70-80heterodimer,

whichformsaringthatencirclestheduplexDNA(GottliebandJackson1993;Ramsden

and Gellert 1998). This protects DNA ends from degradation, and recruits additional

NHEJ components (Fig. 3a). Next, the Ku70-80-bound ends are tethered by DNA-

dependent protein kinase catalytic subunit (DNA-PKcs), followed by ligation of the

broken DNA ends by the XRCC4-XLF complex and DNA Ligase IV. The yeast Mre11-

Rad50-Xrs2(MRX)complexhasastructuralroletopromoteligation,whilethefunction

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ofthehumanMRE11-RAD50-NBS1(MRN)complexinNHEJislessapparent(Chenetal.

2001;HuangandDynan2002;Zhangetal.2007;Rassetal.2009;Xieetal.2009).

InthecaseofDNAendsthatarenotdirectlyligatable,whichmayincludethosewith

DNAoverhangs,gapsorblockingchemicalgroups,limitedDNAendprocessingmaybe

required.This involves anucleolytic removal of overhangsor chemical groupsby the

humanArtemisnuclease,whichcleavesat the junctionsofsingleanddouble-stranded

DNA,andisactivatedbyDNA-PKcs(Maetal.2002;Changetal.2017;LobrichandJeggo

2017). Artemis may not be the only NHEJ nuclease; other proteins, including APLF,

Werner'ssyndromehelicase(WRN),theMRNcomplex,FEN1andEXO1mayalsoplaya

role insomecases(Changetal.2017).Alternatively, the fillingofDNAgapsatbreaks

mayfacilitateligation,whichiscarriedoutbyDNApolymerasesμandλ(Bebeneketal.

2014; Moon et al. 2014). Additionally, polynucleotide kinase (PNK) may remove 3'

phosphategroupsandphosphorylate5'OHgroups,whichmaybenecessaryforligation

(Chappelletal.2002).Finally,onerecentstudyfoundthatextendedDNAendresection

can occur prior toNHEJ in G1 (Biehs et al. 2017), but furthermechanistic analysis is

requiredtofullyunderstandthisprocess.

2.2ProcessingofDNAendforhomologousrecombination

In contrast toNHEJ, extendedDNA end resection is an obligate step that initiates all

recombinationpathways.DNAendresectionofthe5'-terminatedDNAstrandoccursin

two main steps (Mimitou and Symington 2008; Zhu et al. 2008). The first step is

catalyzed by the MRN complex and CtIP in human cells, and MRX and Sae2 in S.

cerevisiae(JohzukaandOgawa1995;KeeneyandKleckner1995;PaullandGellert1998;

Sartorietal.2007).Thenucleolyticprocessingbytheseproteinsislimitedtothevicinity

oftheDNAend(generallyupto300ntinyeast),andisthusreferredtoasshort-range

DNAend resection (Zhuet al. 2008).Themost likelymechanism for the short range-

resectionbyMRX/NisillustratedinFig.5.Resectionisinitiatedbytheendonucleolytic

cleavageof the5'-terminatedDNAstrandaway fromtheDNAend, followedby3'→5'

exonucleasethatproceedsbacktowardtheDNAend.

Both exonuclease and endonuclease activities during the first resection step are

likelycatalyzedbyMRE11/Mre11inhumanandyeastcells(Nealeetal.2005;Garciaet

al. 2011; Cannavo and Cejka 2014; Shibata et al. 2014). MRE11/Mre11 first

endonucleolytically cleaves 5' terminated DNA in the vicinity of the DNA end. This

endonucleolytic cleavage requires the ATPase activity of RAD50/Rad50 as well as

CtIP/Sae2 andNBS1(butnot strictlyXrs2 in yeast) as co-factors (CannavoandCejka

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2014; Anand et al. 2016; Deshpande et al. 2016; Oh et al. 2016; Kim et al. 2017).

Importantly, the capacity of Sae2 and CtIP to promote MRE11/Mre11 depends on

phosphorylationofkeyresiduesinCtIP/Sae2,at leastsomeofwhichareundercyclin-

dependentkinaseCDKcontrol(Huertasetal.2008;HuertasandJackson2009;Cannavo

and Cejka 2014; Anand et al. 2016; Deshpande et al. 2016). Cell-cycle dependent

phosphorylationofCtIP/Sae2representsoneofthekeycontrolmechanismsthatallow

resection(andhencerecombination)toinitiateonlyinSandG2phasesofthecellcycle

when a sister chromatid is available as a template for repair (Orthwein et al. 2015).

Downstream of the endonuclease cut, MRE11/Mre11 subsequently uses its 3'→5'

exonuclease activity to proceed back toward the DNA end, generating a 3' ssDNA

overhang. Additionally, another nuclease, EXD2, may function alongside MRE11

exonuclease in human cells (Broderick et al. 2016). It has been also proposed that

CtIP/Sae2arenucleases(Lengsfeld etal.2007;Makharashvili etal.2014;Wangetal.

2014),buttheirpotentialcatalyticfunctionsinresectionremainundefined.

The initial endonucleolytic cleavage away from theDNAendallows the resection

machinery to bypass end-binding factors or non-canonical structures that may be

presentatthebreakend.Thisincludesproteinblocks,suchasSpo11inmeiosis,stalled

topoisomerasesorKu(KeeneyandKleckner1995;Keeneyetal.1997;Nealeetal.2005;

Bonetti etal. 2010;Mimitou andSymington2010; Langerak etal. 2011;Chanut etal.

2016).Indeed,theefficiencyof5'DNAendcleavageinvitrobyMRN-CtIPorMRX-Sae2is

stimulated by the presence of protein blocks at DNA ends (Cannavo and Cejka 2014;

Anandetal.2016;Deshpandeetal.2016).Theshort-rangeDNAendresectionpathway

is absolutely required for the processing of protein-blocked DNA ends, but may be

dispensable for theresectionofcleanDNAends inyeast (Nealeetal.2005;Zhuetal.

2008;MimitouandSymington2010).Instead,bothMRE11andCtIParerequiredforall

resection events in human cells, although it remains to be definedwhether resection

alwaysdependsonthenucleaseofMRE11(Sartorietal.2007).

The initial endonucleolytic cleavage byMRE11/Mre11 creates entry sites for the

long-range resection enzymes. These subsequently catalyze resection in the 5'→3'

directionawayfromtheDNAendtogenerateextendedssDNAoverhangs(uptoseveral

kilobasesinlength)andrepresentthesecondstepoftheresectionprocess.The3'→5'

exonucleolytic DNA degradation byMRE11/Mre11 and the 5'→3'degradation by the

long-range enzymes downstream of the endonucleolytic cut has been termed "bi-

directional"resection.Inadditiontothenucleasefunction,theMRN/MRXcomplexhas

non-catalytic(i.e.structural)rolestorecruitthelong-rangeresectionenzymes(Cejkaet

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al.2010a;Nicoletteetal.2010;Niuetal.2010;Nimonkaretal.2011).Thelong-range

resectionfactorsincludeeitheroftwonucleases,EXO1/Exo1orDNA2/Dna2,whichare

well conserved between human and yeast cells (Gravel et al. 2008; Mimitou and

Symington2008;Zhuetal.2008).EXO1/Exo1isadsDNAspecificexonuclease(Tranet

al. 2002), which specifically degrades the 5'-terminated DNA strand within dsDNA,

generating 3' ssDNA overhangs. In contrast DNA2/Dna2 is an ssDNA specific 5'→3'

nuclease that cannot process dsDNA on its own, and requires a cognate RecQ family

helicasepartner(Baeetal.1998;Zhuetal.2008;Levikovaetal.2013).This includes

Sgs1 in yeast and either Bloom syndrome helicase (BLM) or WRN in human cells

(Sturzenegger et al. 2014; Pinto et al. 2016; Levikova et al. 2017). Sgs1/BLM/WRN

unwinds dsDNA to generate ssDNA, which becomes rapidly coated by replication

proteinA(RPA).RPA-coatedssDNAissubjecttodegradationbyDNA2/Dna2(Cejkaet

al. 2010a; Niu et al. 2010). RPA was found to promote 5' DNA end degradation by

DNA2/Dna2,whileatthesametimeinhibiting3'enddegradation.RPA,whichphysically

interactswith the RecQ family helicase and DNA2/Dna2, is thus a critical factor that

enforces the correct DNA polarity of DNA end resection by DNA2/Dna2 (Cejka et al.

2010a;Niuetal.2010).BothhumanDNA2andyeastDna2,inadditiontotheiressential

nuclease activity, contain a helicase domain, which likely functions as an ssDNA

translocase to facilitate the degradation of 5'-overhanged DNA by the DNA2/Dna2

nuclease(Levikovaetal.2017;Milleretal.2017).TheindividualsubunitsoftheSgs1-

Dna2-RPA,BLM-DNA2-RPAandWRN-DNA2-RPA complexes stimulate theactivities of

theirpartnerstoformintegratedmolecularresectionmachines(Cejkaetal.2010a;Niu

etal.2010;Pintoetal.2016).

MultipleDNAendresectionmechanismsdescribedabove leadto the formationof

3’-tailed ssDNAcoatedbyRPA.Thekey functionofRPA is toprotect ssDNA from the

actionofnucleasesandpreventtheformationofsecondarystructuresthatmightarise

byself-annealingofssDNA(Wold1997).Aswillbedescribed in thesectionbelow, in

SDSA,BIRandcanonical recombinationalDSBRpathways,RPAmustbereplacedwith

the strand exchange protein RAD51. In contrast, SSA shares the initial DNA end

resectionwithother recombination sub-pathways, but isRAD51-independent. Instead

of using intact dsDNA as a template, SSA functions by annealing the two resected

strandsofDNA,using large stretchesof sequencehomology to forma stable complex

between the two resected broken DNA molecules (Fig. 4a). This is dependent on

RAD52/Rad52,whichhasacapacitytoannealRPA-coatedssDNA.Afterannealing,the

non-complementary sequences are cleavedbyXPF-ERCC1 (Rad1-Rad10 in yeast) and

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anyremaininggapsarefilledandligatedtocompletetherepairoftheDSB(Bardwellet

al.1994;Ivanovetal.1996;Mortensenetal.1996;Shinoharaetal.1998).

Broken DNA molecules signal the presence of DNA damage to the cellular

checkpointmachinery.Generally,DSBsactivatetheATM(Tel1inyeast)kinase.Inboth

systems,MRN/MRX plays a structural role to activate ATM/Tel1 (Carson et al. 2003;

Uzieletal.2003;LeeandPaull2004;LeeandPaull2005).Uponresection,RPA-coated

ssDNA thenactivates theATR-ATRIP(Mec1-Ddc2 in yeast)pathway (ZouandElledge

2003).ATM/Tel1andATR/Mec1sensorsphosphorylatehundredsofproteintargetsat

SQ/TQ motifs, which activate the proper response to DNA damage. This includes

regulation of DNA repair components and checkpoint proteins, leading to cell cycle

arrest and thus providing time for repair (Jackson and Bartek 2009). Additionally,

unsuccessfulrepairandprolongedcellcyclearrestleadtotheactivationofapoptosisin

highereukaryotes(JacksonandBartek2009).

2.3ProcessingofDNAendsforAlt-EJ(MMEJ)

IntheabsenceofthekeycanonicalNHEJfactors,includingKu70-80,DNA-PKcs,XRCC4,

XLFandDNALigaseIV,cellscanstillrepairDSBsthroughanend-joiningeventreferred

toasMMEJ(SimsekandJasin2010;Changetal.2017)(Fig.3b).ThefrequencyofMMEJ

in DSB repair is not very clear yet (Sfeir and Symington 2015), but it has been

establishedthattheprocessismorecommoninhumancellscomparedtoyeast.Oneof

thehallmarksofMMEJistheuseofsharedsequencemicrohomologybetweenthebreak

points,whichisgenerallylimitedto~2-20ntinlength(Changetal.2017).Overlapping

homologies were observed in junction points upon translocations in human cancers

(Stephensetal.2009),suggestingthattheymayariseduetoMMEJ.Currently,onlyafew

factorshavebeenimplicatedinmediatinghumanMMEJ,includingFANCA,PARP1,DNA

LigaseIII,CtIP,DNA2andPolθ(Poltheta,alsoknownasPolQ),butnoneoftheseappear

tobeabsolutelyessentialforMMEJ(Audebertetal.2004;Bennardoetal.2008;Simsek

etal.2011;Howardetal.2015;Mateos-Gomezetal.2015;Mateos-Gomezetal.2017).

The use of microhomology for repair indicates that MMEJ and recombination-based

mechanisms may share elements of DNA end resection. This is also supported by

observationsthatKu70-80inhibitsMMEJ,andKuisknowntobeinhibitoryforDNAend

resection(MimitouandSymington2010).Theendresection factorsCtIPandDNA2 in

humancellshavebeenidentifiedasbeingimportanttopromoteMMEJeventsbutit is

stillunclearifthisisduetoadirectroleinDNAendresection(ZhangandJasin2011;

Howardetal.2015).Likewise,thegeneticrequirementsforMMEJremaintobedefined

13

in yeast. Interestingly, MMEJ was increased in mre11, sae2 and sgs1 exo1 mutants,

indicatingthattheabsenceofthecanonicalDNAendresectionfactorsisnotlimitingfor

MMEJ(Wangetal.2006;Dengetal.2014).Whetheranotheryetunidentifiednucleaseis

essentialforMMEJisnotclear.RPAisclearlyastronginhibitorofMMEJinbothyeast

andhumancells(Dengetal.2014;Mateos-Gomezetal.2017).Thisshowsthatthemore

extensiveresectioncreatingssDNAoflengthsthatstronglyassociatewithRPAchannels

repair towardHR. Inaccord, themotoractivityof humanPolθwas foundtodisplace

RPAtopromoteMMEJ,showingthatthebalancebetweenHRandMMEJiscontrolledby

theopposingactivitiesofPolθandRPA(Mateos-Gomezetal.2017).

3FormationofRAD51-ssDNAfilamentonresectedDNA

Resection of the 5’-terminated DNA strand at DSBs leads to the formation of 3’-

overhanged ssDNA, which is initially coated by RPA. In a subsequent step, RPA is

replacedbythekeyrecombinationproteinRAD51(Rad51inyeast)(Fig.6).RAD51and

ssDNAformanucleoproteinfilament,alsocalledapre-synapticfilament.Thiscatalyzes

the signature step of the recombination pathway, which includes homology search,

pairingwiththeintactdonor(alsocalledtemplate)dsDNAandstrandinvasion(Benson

etal.1994;Sugiyamaetal.1997).ThetransientinteractionoftheRAD51nucleoprotein

filamentwiththetemplatedsDNAisreferredtoasasynapticcomplex.Thisultimately

leads to thedisplacementofoneof the templateDNAstrands formingadisplacement

loop (D-loop, also called joint molecule) intermediate (Fig. 6), which represents the

post-synapticstage.Allthesestepsarecontrolled(bothpositivelyandnegatively)bya

numberof recombination regulators,whichoften physically interactwithRAD51and

allowrecombinationtooccuronlyinthepropercontext.

TounderstandmechanismsunderlyingtheregulationofRAD51,it is importantto

understandthatRAD51hasacapacity tobindbothsingle-anddouble-strandedDNA,

butonlyssDNAbindingisthoughttopromoterecombination,whiledsDNAbindingby

RAD51 is generally inhibitory (Zaitseva et al. 1999). Furthermore, RAD51 binds and

hydrolyzesATP.ATPbinding,butnotATPhydrolysis,byRAD51isrequired forstable

RAD51-DNA binding; in contrast, ATP hydrolysis by RAD51 is required for the steps

downstream of DNA invasion (Sung 1994; Baumann et al. 1996; vanMameren et al.

2009).RAD51canalsohydrolyzeATPnon-productively(i.e.,withoutcatalyzingstrand

invasion);also inthiscase,ATPhydrolysis leads toareduction in itscapacity tobind

DNA. Recombination can be stimulated on several levels by affecting these RAD51

activities.ThisincludesRAD51loadingonRPA-coatedssDNA(i.e.exchangeofRPAwith

RAD51), reduction of RAD51's capacity to bind dsDNA, stabilization of the nascent

14

nucleoproteinfilamentbyinhibitingitsdisassembly,aswellasremodelingoftheRAD51

nucleoproteinfilamentintoaconformationthatisoptimallypermissiveforDNAstrand

exchange. These control mechanisms are in place to prevent aberrant recombination

(Heyer2015).Thisincludesrecombinationbetweennon-homologoussequences,which

mayleadtoDNAtranslocationsandgenomerearrangements,orrecombinationduring

physiological processes of DNA metabolism when ssDNA is present, such as during

unperturbedDNAreplicationortranscription.Theproperinterplayofbothpositiveand

negativerecombinationregulatorsascertainsthatrecombinationoccursonlywhenitis

neededtooptimallymaintaingenomestability(Heyer2015).

The first of the control mechanisms is the replacement of RPA with RAD51 on

resected ssDNA. Due to the higher affinity toward ssDNA, RPA initially outcompetes

RAD51forssDNAbinding.ToovercomethisapparentinhibitoryeffectofRPA,cellshave

variousrecombinationmediatorproteinsthathelploadRAD51ontossDNA,displacing

RPA in theprocess. Inyeast,thekeyrecombinationmediator thatdisplacesRPA from

ssDNA is Rad52. The interaction of Rad51 with Rad52 is required for this process

(Benson et al. 1998; Song and Sung 2000). Human RAD52, in contrast, possesses no

recombinationmediatoractivity,anditscontributiontorecombinationinhumancellsis

much more subtle than in yeast, while Caenorhabditis elegans and Drosophila

melanogaster lackRAD52.Themainmediatorprotein inhigher eukaryotes, including

worms, flies and humans, is the product of the breast cancer susceptibility gene 2

(BRCA2)anditshomologues(Yangetal.2005;Petalcorinetal.2007;Jensenetal.2010).

DepletionofBRCA2incellstreatedwithionizingradiation(IR)leadstopersistentRPA

anddecreasedRAD51fociformationonbrokenDNA,clearlyindicatingitsroleinRAD51

loading(Yuanetal.1999).BRCA2containseightBRCrepeats(conservedmotifsofabout

35aminoacids),whichcanallindependentlyinteractwithRAD51,althoughonlyfourto

five repeats associatewith RAD51 at a given time (Carreira et al. 2009; Jensen et al.

2010).Therepeats1-4bindRAD51withahighaffinityandpromoteRAD51nucleation

on ssDNA; repeats 5-8 show a lower affinity to free RAD51 and rather stimulate the

growthofthenascentRAD51filament(Jensenetal.2010;Liuetal.2010;Thorslundet

al. 2010). Mechanistically, the BRC repeats 1-4 of BRCA2 promote ssDNA binding of

RAD51 by inhibiting its ATPase activity, which stabilizes ssDNA binding of RAD51.

Additionally, BRCA2 promotes recombination by inhibiting RAD51's dsDNA binding

activity(Jensenetal.2010).ThedisplacementofRPAfromssDNAbyBRCA2isfurther

facilitatedbyDSS1,adirectinteractionpartnerofBRCA2(Yangetal.2002;Zhaoetal.

2015). BRCA2 also interacts with PALB2, which was shown to promote RAD51-

mediated DNA strand exchange on its own (Buisson et al. 2010). In cells, PALB2

15

mediates BRCA2's recruitment to DNA damage and bridges BRCA2's interactionwith

BRCA1(Syetal.2009).Cellularassaysestablishedthattheseinteractionsarecriticalfor

recombination, although the mechanisms how BRCA1 and PALB2 proteins affect

BRCA2's recombinationmediator activity remain to be defined. RAD51 nucleoprotein

filament assembly is also stimulated by RAD54, independently of RAD54's ATPase

activity(WolnerandPeterson2005).AdditionalproteinsincludingtheMMS22L-TONSL

complexmay promote the RAD51 nucleoprotein filament assembly during perturbed

DNAreplication(Piwkoetal.2016).

The activity of RAD51 is also promoted by a group of proteins termed RAD51

paralogs.Therespectivegeneslikelyaroseduringevolutionthroughaduplicationofthe

RAD51 gene, and share around 20 to 30% of sequence homology with RAD51. The

paralogs are represented by five polypeptides in human cells: RAD51B, RAD51C,

RAD51D, XRCC2 and XRCC3, which form two major complexes: RAD51B-RAD51C-

RAD51D-XRCC2(BCDX2)andRAD51C-XRCC3(CX3)(Massonetal.2001).Neitherofthe

paralogproteinsnorcomplexesexhibitsDNAstrandexchangeactivityontheirown,and

all likely function via regulatingRAD51. Phenotypically, depletion of these complexes

generallyresultsinfewerRAD51fociinresponsetoionizingradiation.Thisresembles,

althoughtoalesserextent,thedepletionofBRCA2.Theparalogslikelyfunctioninthe

samepathwayasBRCA2as indicatedby epistatic interactions(Qing et al. 2011).The

first mechanistic report indicated that the RAD51B-RAD51C proteins, similarly to

BRCA2,havearecombinationmediatoractivitytofacilitateloadingofRAD51onRPA-

coated ssDNA, displacing RPA in the process (Sigurdsson et al. 2001). In yeast, the

RAD51paralogsincludeRad55andRad57(Sung1997;Liuetal.2011a).Rad55-Rad57

physically interact with the Shu complex, comprising of Csm2, Psy3, Shu1 and Shu2

polypeptides,andRad52.Thesubunitsofthesupercomplexsynergizeintheircapacity

todisplaceRPAandfacilitateRad51-ssDNAnucleoproteinfilamentformation(Gaineset

al.2015).ThehumanorthologofShu2 isSWS1,which formsacomplexwithSWSAP1

(Martin et al. 2006; Liu et al. 2011b).TheheterodimerbindsDNAand interactswith

RAD51,RAD51DandXRCC2. DepletionofSWS1resultsinareductionofspontaneous

and radiation-induced RAD51 foci. This suggests that SWS1 in humans also likely

functions inconjunctionwith theRAD51paralogs,but their interplay,aswellas their

relationshiptoBRCA2'sfunction,remainundefined.EvidencefromtheC.elegansmodel

system instead suggests that the RAD51 paralogs, represented by RFS-1 and RIP-1,

function downstream of BRCA2, specifically in the stabilization of the RAD51

nucleoprotein filament and its remodeling into a species that is optimally capable to

16

invadetemplatedsDNA(Tayloretal.2015;Tayloretal.2016).RAD51filamentsarealso

stabilizedbytheSWI5-SFR1complex(AkamatsuandJasin2010;Tsaietal.2012).

It is well established that there is a balance between factors that promote the

formation and disruptRAD51/Rad51 nucleoprotein filaments (Heyer 2015). In yeast,

Srs2candismantleRad51 filamentsdue to itsATP-poweredDNAtranslocaseactivity,

andadditionallyviastimulatingtheATPhydrolysisofRad51throughadirectphysical

interaction,whichdestabilizesthenucleoproteinfilament(Krejcietal.2003;Veauteet

al.2003;Liuetal.2011a).Inhumans,nodirecthomologofSrs2hasbeenidentified,yet

themostprobablecandidatesforthisfunctionareRECQ5,FBH1,PARIorBLMhelicases

(Bugreev et al. 2007b; Hu et al. 2007; Fugger et al. 2009; Schwendener et al. 2010;

Moldovan et al. 2012; Patel et al. 2017). To elucidate the functional interplay of the

various RAD51 regulators represents a challenge for future research. The proper

balance between pro- and anti-recombination factors is required to execute

recombinationonlywhenitisneededandthuspreventillegitimaterecombinationand

genomerearrangements.

4HomologysearchandDNAstrandexchange

Once the pre-synaptic filament is formed and stabilized, it begins the search for a

homologoussequence.Inmostcasesinvegetativecells,thesisterchromatidisusedasa

repairtemplate.ArecentreportdemonstratedthatthehumanBRCA1protein,whichis

similarlyasBRCA2atumorsuppressorthatisfrequentlymutatedinfamiliarbreastand

ovariancancers,directlypromotesDNAinvasion.Thislikelyoccursthroughpromoting

the assembly of the synaptic complex or promoting the homology search (Zhao et al.

2017).Despitethecomplexnuclearenvironment,thecourseofhomologysearch–the

processthatoccursimmediatelybeforethesynapticphase–isrelativelyfast.Inyeast,it

has been demonstrated that the mobility of a cut chromosome is increased, which

allowstheRad51filamentstoexplorealargernuclearvolume(Dionetal.2012;Mine-

Hattab and Rothstein 2012). Additionally, broken DNA relocalizes to the nuclear

periphery(Ozaetal.2009;Chioloetal.2011;Horigomeetal.2014;Ryuetal.2015).

Althoughtheexactmechanismofhomologysearchisstillundefined,itissuggestedthat

thepre-synapticfilamentrandomlyprobesthegenomebymakingmultipletemporary

contactswithdifferentDNAduplexes(ForgetandKowalczykowski2012;Renkawitzet

al. 2014; Qi et al. 2015). Contacts with very short microhomologies are unstable;

instead, contactswithmore than7nt of homology aremore stable,which allows the

presynaptic complex to probe flanking sequences for additional homology. Once

17

homologyisidentified,thepre-synapticfilamentinvadestheduplexDNA,displacesthe

originalstrandandbinds itscomplementarysequencebyWatson-Crickpairing. Ithas

been estimated that the efficiency of repair in yeast was decreased to ~14% when

templatesequencedivergedbyaboutoneineveryeightnucleotides(Anandetal.2017).

Followingstrandinvasion,thedisplacedssDNAwithintheD-loopstructureisstabilized

byRPA,whichpreventsreversaloftheD-loopformation(LaveryandKowalczykowski

1992;Eggleretal.2002).Additionally,RAD51isremovedfromtheheteroduplexdsDNA

oftheD-loopstructure.ThisfunctioniscatalyzedbyRAD54,adsDNAtranslocasethat

usesitsATPase-poweredmotoractivitytodisplaceRAD51fromdsDNA(Solingeretal.

2002;Masonetal.2015)(Fig.6).RAD54thuspromotesD-loopstability,whichallows

DNAsynthesisandfacilitatescanonicalHR(CeballosandHeyer2011;WrightandHeyer

2014).

The 3'-terminated strands within D-loop structures that resist disassembly can

prime DNA synthesis. Although translesion polymerases have been implicated in HR

(Kawamoto et al. 2005; McIlwraith et al. 2005), most of DNA synthesis during

recombinationislikelycatalyzedbyeitherpolymeraseδorpolymeraseε(Lietal.2009;

Hicksetal.2010;Wilsonetal.2013).However,theDNAsynthesisduringrecombination

is about three orders of magnitude more error-prone than in DNA replication, most

likelyduetolimitedactivityofDNAmismatchrepairduringrecombination-dependent

repairDNAsynthesis(Hicksetal.2010).DNAsynthesisextendsthelengthofthepaired

duplex, which further stabilizes the joint molecule, leading to the recovery of the

missinggeneticinformationinthebrokenDNAmoleculebyusingtheinvadedmolecule

as a template. Downstream of strand invasion and DNA synthesis, recombination

proceedsintoeitherofthreerecombinationsub-pathways:BIR,SDSAandcanonicalHR

(see chapter 1.3 for general introduction to these pathways and Fig. 4). In BIR, the

invadedDNAmoleculeisstabilized,andDNAsynthesisproceedsalongthewholelength

ofthetemplateDNAviaabubble-likestructuretothechromosomeend(Llorenteetal.

2008;SakofskyandMalkova2017).TheextendedDNAsynthesisislikelycatalyzedby

polymerase δ, in conjunction with the Pif1 helicase, which promotes the strand

displacementactivityofpolymeraseδ(Wilsonetal.2013).Interestingly,DNAsynthesis

during BIR occurs conservatively, and thus dramatically differs from canonical DNA

replication (Donnianni and Symington 2013). How exactly the synthesis of the

complementarystrand isachieved remainsundefined.TheBIRpathwayoccurs at the

cost of elevated mutagenesis, and is thus primarily utilized when no alternative is

available,suchasintheabsenceofthesecondDNAend.

18

InSDSA, theextended invadedDNAstrandseparates fromthe templateDNA,and

annealstothesecondendofthebrokenDNAmolecule.DNAsynthesisandligationthen

completetherepairprocess.IncanonicalHRinstead,throughaprocesstermedsecond

DNAendcapture,thesecondbrokenDNAendannealstothedisplacedssDNAstrandof

theD-loopstructure(Fig.4).ThestrandannealingemploystheRad52proteininyeast

(Nimonkar et al. 2009). Although humanRAD52 also possesses the strand annealing

activity(McIlwraithandWest2008;Jensenetal.2010),theeffectsofRAD52-deficiency

are modest and are particularly revealed only in the absence of BRCA2 (Feng et al.

2011).Thisraisesquestionswhetherthesecondendcaptureinhumancellsismediated

byannealingorasecondDNAstrandinvasionevent(Kowalczykowski2015).

The balance between SDSA and canonical recombinational DSBR is regulated by

activitiesthateitherdisruptorpromotestabilityoftheD-loopstructure.Inmostcases,

thestabilityofD-loopsisdictatedbymotorproteinssuchasDNAhelicasesthatactby

moving the junctions.Mph1 inyeastcellsandBLM,RECQ1andRTEL1 inhumancells

have been implicated in D-loop dissociation and promotion of SDSA (Bugreev et al.

2007b;Barberetal.2008;Bugreevetal.2008;Prakashetal.2009;Daleyetal.2013;

Mitchel et al. 2013). In yeast, D-loops can also be disrupted or "dissolved" by an

alternativemechanismthatinvolvestopoisomeraseIII(Top3),whichisalsothoughtto

promoteSDSA(Faschingetal.2015).Whetherthisprocessalsofunctionsinhumancells

isnotyetclear.Additionally,RAD54'sbranchmigrationactivityhasbeenimplicatedto

disruptD-loopsdownstreamofRAD51-mediatedstrandinvasioninvitro(Bugreevetal.

2007a), but the biological significance of this function may be restricted to limiting

recombinationwithnon-homologousDNA(CeballosandHeyer2011;WrightandHeyer

2014). Regulating the balance between SDSA and HR is important, as it affects the

geneticoutcomeofrecombination.WhereasSDSAonlyleadstonon-crossoverproducts,

canonicalHRisapathwaythatcanpotentiallyproducecrossovers,aswillbedescribed

inthenextchapter.

5Processingrecombinationintermediates

D-loop stability determines the pathway choice between SDSA and canonical

recombinational DSBR. In SDSA, the D-loop is disrupted, whereas in recombinational

DSBR, theD-loop isstabilizedandbecomesasubstrate forannealingwith thesecond

resecteddsDNAend(Fig.4).Thisgivesrisetoa"double"or"complement-stabilized"D-

loop.ThisstructureformsasaresultofannealingactivitycatalyzedbyRad52inyeast,

and doesnot represent a second strand invasion step (Nimonkar et al. 2009). Rad52

19

wasshowntopromoteannealingofssDNAtoD-loopsgeneratedbycognateRad51and

Rad54inthepresenceofRPA,indicatingthattheprocesslikelyrequiresdirectprotein-

proteininteractions.ThisfunctionofRad52isapparentinunicellulareukaryotes,while

RAD52inhumancellshasamuchlessdefinedfunction(Fengetal.2011).Itispossible

thatinhigheukaryotesotherproteinssuchasBRCA2mightbeinvolvedinsecondend

captureinadditiontoRAD52.Itremainstobeestablishedwhethersecondendcapture

inhumancellsemploysannealingand/orstrandinvasionmechanisms.

Following second end capture, DNA synthesis and ligation gives rise to a central

intermediate of canonical HR, termed a double Holliday junction (dHJ)(Duckett et al.

1988).AsbothDNAmoleculesarephysicallylinkedatthejunctionpoints,HJsneedto

beprocessedpriortoseparationofbothDNAmolecules(Fig.7).AfailuretoprocessHJs

leads to chromosome segregation defects, and may be one of the mechanisms

responsibleforgenomeinstability(Wechsleretal.2011).Duetothehomologybetween

therecombiningDNAmolecules,akeyfeatureofendogenousHJsistheirmobility:the

junctionpointscanmoveineitherdirectiontoalimitedextentspontaneously,ormore

extensively inATP-hydrolysisdriven reactions catalyzedbymolecularmachines, such

as DNA helicases or translocases. Double HJs can be processed by resolution or

dissolution-based mechanisms. As will be described below, these processes are

enzymaticallydistinctandleadtodiversegeneticoutcomes.Whereasdissolutionleads

tonon-crossoverproducts,resolutiongivesrisetobothcrossoversandnon-crossovers

(Fig.4andFig.7).

5.1DissolutionofdoubleHollidayjunctions

DissolutionseparatestherecombiningDNAmoleculeswithoutexchangingtheflanking

sequences. As somatic cells employ mechanisms to maximally preserve genome

integrity, dissolution is the default mode of dHJ processing. In yeast, dissolution is

carriedoutbytheSgs1-Top3-Rmi1(STR)complex(Cejkaetal.2010b),whileinhuman

cells the"dissolvasome"consistsofBLM, topoisomerase IIIαandRMI1-RMI2, forming

theBTRRcomplex(WuandHickson2003;Singhetal.2008;Xueetal.2013).AsSgs1,

BLM can unwind various DNA structures and branch migrate HJs in vitro (Wu and

Hickson 2003). The dissolution reaction involves migration of the two Holliday

junctionstowardeachother(i.e.convergentbranchmigration)bythecombinedactivity

of the RecQ family BLM/Sgs1 helicase and the strand passage activity of the

TOPOIIIα/Top3 type IA topoisomerase (Fig. 7a). As both Holliday junctions link two

DNAmoleculesoflonglengths,theendsarenotfreetorotate,andthedHJstructureis

20

topologically constrained. Therefore, a branch migration activity of Sgs1/BLM is not

sufficienttomigrateendogenousHJs(Cejkaetal.2010b;Chenetal.2014).Ithasbeen

demonstratedthatthestrandpassageactivityofTOPOIIIα/Top3isrequiredtorelieve

positive supercoiling thatwouldotherwise formaheadof (between) the convergently

migratingjunctionsandpreventfurthermovement(Chenetal.2014).Mechanistically,

Top3/TOPOIIIαcreatesatransientnickinssDNAthatallowstheotherssDNAstrandto

passthroughit,therebyallowingtherelaxationofthetorsionalstressformingbetween

the junctions during convergent branchmigration. RMI1/Rmi1 does not significantly

affect the initial DNA branchmigration step, but seems to specificallypromote a late

stepjustpriortodissolution(Cejkaetal.2010b;Bocquetetal.2014).Thelastpredicted

intermediate of convergent branch migration is a hemicatenane: this structure

representsajunctionbetweentwodsDNAmolecules,whereonestrandofoneduplexis

wrapped around another strand from the second DNA duplex (Fig. 7a). It has been

proposed that RMI1/Rmi1 specifically promotes the processing of a hemicatenane

(Cejka et al. 2012). RMI2,which is onlypresent inhigh eukaryotes, likelyhasonly a

minor function indHJdissolution,andmayhaveother roles such as to targetBLMto

blocked DNA replication forks (Singh et al. 2008). Finally RPA, which physically

interactswith BLM/Sgs1 and RMI1/Rmi1 subunits of the STR/BTRR complexes, also

stimulatesdissolution(Cejkaetal.2010b;Xueetal.2013).ThislikelystemsfromRPA's

capacity to promote the helicase of BLM/Sgs1 and strand passage of TOPOIIIα-

RMI1/Top3-Rmi1, likelythroughitsssDNAbindingactivitytopreventre-annealing,as

wellasbecauseofthedirectphysicalinteractionofRPAwithRMI1/Rmi1(Broshetal.

2000;Planketal.2006;Cejkaetal.2012;Xueetal.2013).TheuniquemechanismofdHJ

processingbydissolutionexclusivelyresultsinnon-crossoverevents,whichmaintains

genome stability in vegetative cells (Fig. 7a). BLM-deficient cells are characterized by

elevated levels of sister chromatid exchanges, which are a hallmark of genome

rearrangements and may contribute to cancer predisposition of Bloom syndrome

patients (Chaganti et al. 1974). These rearrangements result from elevated usage of

pathways thatare alternative todissolutionand thataredependenton the structure-

specificnucleasesdescribedinthenextchapter.

5.2ResolutionofdoubleHollidayjunctions

DoubleHJs thatevadedissolutionareresolvedbystructurespecificnucleases later in

thecellcycletogiverisetobothcrossoverandnon-crossoverrecombinationproducts.

Nucleolytic cleavage also represents the only option for the processing of singleHJs.

21

Thesemay form duringDSB repairwhere only one strand invades template DNA, or

whenoneoftheD-looparmshasbeencleavedpriortosecondendcapture(Wechsleret

al.2011;ShahPunataretal.2017).Todate,threestructure-specificnucleasescapableof

processingHJsorsimilarstructureshavebeen identified ineukaryoticcells, including

humanMUS81-EME1andyeastMus81-Mms4,humanSLX1-SLX4andyeastSlx1-Slx4as

well as human GEN1 and yeast Yen1 (Dehe and Gaillard 2017). The Mus81-Mms4

complex in yeast was found to be essential for viability in cells lacking Sgs1-Top3,

indicating its role in the processing of recombination intermediates thatwould have

otherwise been processed by the Sgs1-Top3 complex (Hickson andMankouri 2011).

Mus81belongstotheXPFfamilyofnucleasesandrepresentsthecatalyticsubunitofthe

heterodimer. Both yeast and humanMus81-Mms4/MUS81-EME1 complexes prefer to

cleavereplicationforks,3’flapsandnickedHJs(Gaillardetal.2003;EhmsenandHeyer

2008). MUS81-EME1/Mus81-Mms4 cleave intact HJs inefficiently in an asymmetric

manner by introducing a nick a few nucleotides away from the junction, producing

gapped and flapped DNA products, which are unsuitable for ligation and necessitate

furtherprocessing(Wyattetal.2013). Inhumancells,MUS81 formsanothercomplex

withEME2,whichhasabroadersubstratespecificitythanMUS81-EME1,andhasroles

in replication fork restart (Pepe andWest 2014b). SLX1/Slx1 belongs to the GYI-YIG

familyofnucleasesandisthecatalyticsubunitoftheSLX1-SLX4heterodimer.TheSLX1-

SLX4/Slx1-Slx4 complex can cleave branched structures with a preference toward Y-

structures,5' flapsandreplication forks.While itcancleave intactHJs, thecleavage is

inefficient,asymmetrical and createspoorly ligatableproducts (Fricke andBrill2003;

Fekairi et al. 2009; Svendsen et al. 2009;Wyatt et al. 2013). BothMUS81 and SLX1

complexes are thus non-canonical HJ resolvases. The only bona-fide eukaryotic HJ

resolvase is GEN1/Yen1,which belongs to the RAD2/XPG family of nucleases. As the

EscherichiacoliRuvC,GEN1/Yen1dimerizesonHJs,andcleavesHJsbyadualincision

mechanismthatproducestwoligatableproducts(Dunderdaleetal.1991;Ipetal.2008;

Rassetal.2010).However,bothGEN1andYen1arecapableofcleavingotherbranched

DNA structures as well. The random nature of HJ cleavage by the various nuclease

complexes gives rise to both crossover and non-crossover recombination products

(ShahPunataretal.2017)(Fig.7).

Inhumancells,boththeSLX1-SLX4andMUS81-EME1dimersassociatetoformthe

SLX1-SLX4-MUS81-EME1 tetramer(SMcomplex).The formationof the SMcomplex is

mediatedbytheinteractionbetweenMUS81andSLX4polypeptides(Wyattetal.2013).

Biochemical studies demonstrated that the SM complex exhibits higher HJ resolution

activitycomparedtoitsindividualsubunits.Mechanistically,SLX1-SLX4makesthefirst

22

nick in the HJ creating a substrate for MUS81-EME1, which then makes a second

counter-nick in the opposing strand, allowing for efficientHJ resolution (Castor et al.

2013;Wyatt et al. 2013).This activity is further enhancedby interactionwitha third

nucleasecomplex,theXPF-ERCC1dimer,whichhasastructural(i.e.non-catalytic)role

to promoteHJ cleavage (Wyatt et al. 2017)(Fig. 7b). The catalytic subunitwithin the

XPF–ERCC1dimerisXPF,whichhasestablishedfunctionsinnucleotideexcisionrepair.

It should be pointed out that the functions ofMUS81-EME1/Mus81-Mms4and SLX1-

SLX4/Slx1-Slx4arenotspecifictoprocessingrecombinationproducts,andbothenzyme

complexesmaycleavestructuresarisingduringotherDNArepairprocesses,aswellas

during replication or telomere maintenance. Likewise, the SLX1-SLX4-MUS81-EME1-

XPF-ERCC1 (SMX) trinuclease complex has a broad substrate specificity that is not

restricted to HJ cleavage and likely functions in multiple DNA metabolic pathways

(Wyattetal.2017).Theformationofthenucleasecomplexesappearsspecifictohigher

eukaryotes,asnosuchcollaborativeactionofnucleaseshasbeenobservedinyeast.

5.3RegulationofHollidayjunctionprocessing

To maximally preserve genome stability, vegetative cells evolved mechanisms that

facilitatedissolutionpathwayusageoverresolution,which limitscrossover formation.

Additionally,theactivityofthenucleasescapableofcleavingbranchedDNAstructures

must be carefully controlled to avoid promiscuous DNA cleavage. The preferential

employmentofdissolutionoverresolutionbystructure-specificnucleases isgoverned

bytightspatialandtemporalcontrol(MatosandWest2014).TheSTR/BTRRcomplexis

likelyactiveinanyphaseofthecellcycle,andthusprocessesthemajorityofdHJsfrom

S-phasetomitosisinbothyeastandhumancells.TheactivityofyeastMus81-Mms4is

insteadlowinS-phase,andbecomeselevatedattheonsetofmitosisbyphosphorylation

oftheMms4subunitbyCDKandCdc5(Matosetal.2011;Matosetal.2013).Inhuman

cells, the phosphorylation of EME1 does not activate MUS81 directly, but rather

promotes the formationof the SLX1-SLX4-MUS81-EME1 (SM) complexwith increased

activitytowardHJs(Wyattetal.2013).YeastYen1isinactiveinitsphosphorylatedstate

inSphase,andbecomesactivatedbyCdc14-mediateddephosphorylationlateinmitosis.

Interestingly,phosphorylationofYen1notonlyinhibitsitscatalyticactivitybylimiting

itsassociationwithDNA,butalsopreventsnuclearimport(Kosugietal.2009;Blancoet

al.2014).Nuclearexclusionappearstobetheprimarymechanismthatrestrictshuman

GEN1 activity (Chan and West 2014). The nuclear export sequence within GEN1

ascertains thatGEN1canprocessrecombination intermediatesonlywhenthenuclear

23

envelope breaks down during mitosis. These regulatory mechanisms collectively

ascertainthatthestructure-specificnucleasesareonlyactivatedlateinthecellcycleto

removeany residual junctions thatwerenotprocessedby thedissolvasome complex.

Althoughpotentiallymutagenic,efficientprocessingofalljointmoleculesbyresolution

mechanismsisessentialtopreventchromosomemissegregationinmitosis(Wechsleret

al.2011).

6SpecializedrolesofDSBrepairpathways

CellsevolvedmechanismstorepairaccidentalDNAbreakstoachievemaximalefficiency

and accuracy in the maintenance of genome integrity. However, not all DSBs are

pathological,andthereareseveralcaseswhenDSBsareintroduceddeliberately,which

serves specific physiological purposes. The best examples are processes occurring

during lymphocyte development and inmeiosis. Aswill be seen below, during these

events cellsmakeuseof theDSB repairpathways to insteadgeneratediversity.Both

processesrepresentfascinatingexamplesoftheplasticityoftheDSBrepairsystems.

6.1DSBrepairpathwaysintheimmunesystem

V(D)JrecombinationisaprocessthatoccursduringBandTlymphocytedevelopment

andinvolvesarandomrearrangementofthevariable(V),diversity(D)andjoining(J)

segments of immunoglobulin genes (Arya and Bassing 2017) (Fig. 8a). The random

assemblyof theseVDJ segmentsallowsproducingawidevarietyof antigen receptors

from a limited number of gene segments. Despite its name, V(D)J recombination is

facilitatedbycanonicalNHEJfactors,anddoesnotinvolvehomologousrecombination.

DisruptionofkeyNHEJfactorsresultsinsevereimmunedisordersinhumansandmice,

indicating theiressentialrole inV(D)Jrecombination.TheMRNcomplexwas foundto

haveafunctioninV(D)Jrecombination,butthisappearstobedependentonitscapacity

topromotecheckpointsignalingviaactivationoftheATMkinase,andnotaroleinHR

(Uzieletal.2003;Helminketal.2009).

V(D)J recombination is initiated by the recombination-activating genes 1 and 2

(RAG1 and RAG2), which are expressed in developing lymphocytes and therefore

restrict this process to these cells (McBlane et al. 1995; Schatz and Swanson 2011).

RAGs bind to the recombination signal sequences (RSS) flanking the V, D, and J gene

segmentsandfirstcreateanssDNAnickatthejunctionbetweenthecodingregionand

theRSS.TherearetwotypesofRSsequences,whichdifferwithrespecttothelengthofa

spacerregionbetweenthetwoidenticalsequences.Thespacersareof12or23bpsin

24

length, giving rise to RS12 and RS23 sequences (Fig. 8a). Next, RAGs catalyze the

formationofapairedcomplex,wheretheRS12andRS23sequencesassociatewiththe

same RAG complex. The free hydroxyl group of the nicked strand then invades the

phosphodiester bond of the intact strand, generating a DSBwithdifferent DNA ends.

ThecodingendoftheDSBcontainsahairpinloop,whereasthesignalendisblunt.Next,

the blunt signal ends are ligated together to form a circular piece of DNA that is lost

duringsubsequentcelldivisions.Thehairpincodingendsareprocessedandjoinedby

theNHEJmachinerytofusetherespectivegenesegments.First,Ku70-80andDNA-PKcs

bind to the coding ends and recruit other additional NHEJ factors, includingArtemis,

XRCC4-XLF andDNA ligase IV (Fig. 8a). Next, the Artemis nuclease is activated upon

autophosphorylationofDNA-PKcsandopensthehairpinloopofthecodingends,which

can then be ligated to the other coding ends by the XRCC4-XLF and DNA ligase IV

complex(Lietal.1995;Casellasetal.1998;Maetal.2002).ThecreationofDSBswith

different ends andthe requirement for associationof the12-and23-RS sequences in

human B and T cells is essential for regulated processing to ensure that coding

sequencesofV,DandJ,butnothomotypicgenesegmentsarejoined.Thefinalproduct

ofV(D)JrecombinationisaDNAcodingsequenceconsistingofrandomlyrearrangedV,

DandJgenesegments(SchatzandSwanson2011).

Classswitchrecombination(CSR)andsomatichypermutation(SHM)onlyoccurin

activatedgerminalcenterBcells.CSRleadstothechangeofantibodyisotypeandthus

itseffectorfunction,whileSHMaffectsthevariableregionsoftheimmunoglobulingenes

to promote diversity of antibodies. Both CSR and SHM are independent of RAGs and

insteadaretriggeredbyactivation-inducedcytidinedeaminase(AID)(Muramatsuetal.

2000; Arakawa et al. 2002; Petersen-Mahrt et al. 2002)(Fig. 8b). AID specifically

deaminatescytosinesintouracils,resultingintheformationofU:Gmismatches.During

CSR,theCμexonisexchangedbyCγ/ε/αexons,resultinginthechangeoftheantibody

isotype from IgM to IgG, IgEor IgA (Methot andDiNoia2017).UnlikeRAGs thatare

targeted to very specific sequences, AID in CSR is active on large regions (1-3 kb) of

repetitive DNA sequences upstream of CH genes (coding for constant regions of

immunoglobulinheavy chains), called the switch(S) regions.TheU:Gmismatchesare

mainly processed by the base excision repairmachinery (BER),which creates ssDNA

breaks as intermediates of the repair process (Rada et al. 2002; Imai et al. 2003;

Schrader et al. 2005). Closely spaced ssDNA breaks then give rise to staggered DSBs

(DSBswithssDNAoverhangs)(Fig. 8b).Additionally, a smaller fractionofDNAbreaks

mayformasaresultofanon-canonicalfunctionoftheDNAmismatchrepairmachinery,

which involves the exonuclease activity of Exo1 (Ehrenstein and Neuberger 1999;

25

Schraderetal.2007;Bregenhornetal.2016).TheexonucleolyticprocessingofoneDNA

strandthencollideswithanickintheoppositeDNAstrand, leadingtoDSBs(Fig.8b).

Irrespectively of the exact mechanism, the resulting DSBs often contain ssDNA

overhangs,whichneed tobe either filledor cleavedprior to joiningby the canonical

NHEJmachinery.DSBswithoverhangsbearingmicrohomologiesmayalsobejoinedby

theMMEJpathway(Lee-Theilenetal.2011).AswithV(D)Jrecombination,CSR isalso

dependentonend-joiningmechanisms,andit isthusnotahomologousrecombination

process.Interestingly,MRNwasfoundimportantforCSR,whereitlikelyhasastructural

roletopromotebothcanonicalNHEJandMMEJpathways(Dinkelmannetal.2009).

Somatichypermutationis,asCSR,dependentonAID(Arakawaetal.2002;Phamet

al.2003).IncontrasttoCSR,SHMleadstoantibodydiversificationthroughmutagenesis

intheVregionsoflightandheavychainsofimmunoglobulins.Ithasbeenestimatedthat

themutation rate in theV regionsduringSHMisabout6ordersofmagnitudehigher

thanintherestofthegenome.Themajorityofmutationsaresinglebasesubstitutions,

withasmallfractionofshortinsertionsordeletions.Thissuggeststhattheformationof

a DSB is not an obligate step in SHM, and in fact NHEJ-deficient cells do not show

significant defects in SHM.However, some DSBs form during SHM (Papavasiliou and

Schatz 2000). To this point, it has been demonstrated that DSBs during SHM can be

repairedbyhomologousrecombination(PapavasiliouandSchatz2000;Zanetal.2003);

furthermore, HR has been implicated as a safeguard against off target AID activity

(Hashametal.2010;Zahnetal.2014).Interestingly,MRNwasfoundtopromoteSHM,

buttheunderlyingmechanismsremainunclear(Yabukietal.2005).

Finally,theinsertionofDNAfragmentsofvarioussizeshasbeenrecentlyreported

to occur in the switch region of immunoglobulin genes (Tan et al. 2016; Pieper et al.

2017).TheDNAsequencesoriginatefromanotherpartofthegenomeandthetransfer

occurs via a copy-paste rather than a cut-paste process. Thismay represent a novel

mechanismforantibodydiversification,althoughthemolecularpathwaysrequiredfor

thesetransactionsareunknown.Interestingly,insertionsoffragmentsfromthecollagen

receptorLAIR1intoimmunoglobulingenesleadtoantibodiesthatarebroadlyreactive

tomalaria(Tanetal.2016;Pieperetal.2017).ItisimportanttopointoutthatduringB

cell maturation, clones that produce high affinity antibodies are positively selected

while lowaffinityclonesareeliminated.Due to theselectionprocess,even infrequent

eventsmaythusbecomephysiologicallyrelevant(Pieperetal.2017).

6.2Homologousrecombinationinmeiosis

26

Meiosisisaspecializedcelldivisionthatisrequiredforgenomehaploidization(Keeney

et al. 2014). This is essential to form spores or gametes in sexually reproducing

unicellularormulticellulareukaryotes,andmakessure thatploidy ismaintainedwith

each successive generation (Fig. 9). DSBs are introduced duringmeiosis, and become

substrates for the homologous recombination machinery (Sun et al. 1989; Sun et al.

1991). The function ofmeiotic recombination inmost organisms including yeast and

vertebrates (but notDrosophila and Caenorhabditis) is to make physical connections

betweenhomologouschromosomestofacilitatetheirproperalignmentandsubsequent

segregation.Additionally, recombinationcreatesgeneticdiversity inthepopulationby

exchanging DNA regions between maternal and paternal chromosomes. The key

mechanisticdifferencesbetweenHRinvegetativecellsandduringmeiosisare(a) the

formationofDNAbreaks,whichareinducedinaprogrammedmannerduringmeiosis;

(b)thepreferentialusageofthehomologouschromosomeasatemplateforrepairand

(c)abiasforpreferentialresolutionofrecombinationintermediatesintocrossoversthat

arenon-randomlyspaced(Fig.9)(Hunter2015).

DSBs are introduced during the first meiotic division by the SPO11/Spo11

transesterase,which is evolutionarily conserved fromyeast tomammals (Keeneyand

Kleckner1995;Keeneyetal.1997;Mahadevaiahetal.2001;Robertetal.2016).SPO11

is a topoisomerase-like protein that cleaves both strands of dsDNA, but remains

covalentlyattachedtothe5'-terminatedDNAstranduponcleavage.Inyeast,atleast9

other proteins are required for Spo11 to cleave DNA, including the MRX complex

(Keeney 2008). Interestingly, the nuclease activity of MRE11 is dispensable for DNA

cleavagebySpo11inyeast,butitisinsteadessentialtoinitiatetheprocessingofSpo11-

boundDNAbreaks(Bordeetal.2004;LamandKeeney2014).Aswithotherprotein-

blocked DNA ends, the first resection step requires the nuclease activity of the

MRN/MRX complex with its co-factor CtIP/Sae2 to remove SPO11/Spo11 from the

breakends(KeeneyandKleckner1995;Nealeetal.2005).ResectionbyMRX-Sae2can

proceeduptoseveralhundredofnucleotidesinlength.Thisisfollowedbylong-range

resection, which appears to be exclusively carried out by the Exo1 branch in yeast

meiotic cells (Zakharyevich et al. 2010; Mimitou et al. 2017). In mice, the average

resectionlengthis0.9kb(Langeetal.2016),whichissimilartothevalueobtainedin

yeast(Zakharyevichetal.2010).

The resected3’DNA tail is boundbyRPAand successively replacedbyDMC1(a

meiosis specific strand exchange protein) together with RAD51 (Bishop et al. 1992;

Cloud et al. 2012).Whereas RAD51 is the only strand exchangeprotein in vegetative

cells, meiotic cells employ both RAD51 and DMC1. In addition to the recombination

27

mediatorssuchasyeastRad52andhumanBRCA2thatfunctionduringrecombinationin

bothvegetativecellsand inmeiosis,meioticcellsmakeuseofanadditionalregulator,

the HOP2-MND1 complex (Leu et al. 1998; Petukhova et al. 2005). The heterodimer

stabilizestheRAD51/DMC1complexonssDNA,reducestheaffinityofRAD51todsDNA

andmodulatestheconformationofthenucleoproteinfilamenttopromoteDNAstrand

exchange(Chietal.2007;Pezzaetal.2007).

ThesecondkeydissimilaritybetweenmeioticandmitoticHRisthetemplatechoice

forrepair.Meioticcellspreferentiallyuse thehomologouschromosome insteadof the

sister chromatid in order to fulfill the requirement to generate genetic variability

(Schwacha and Kleckner 1994; Baudat et al. 2000; Peoples et al. 2002). How this is

achievedisnotyetfullyunderstood.Thetemplatebiasisregulatedbycohesion(Kimet

al. 2010), depends inpart on the role of DMC1 to promote strand exchange between

homologs, and requires the DNA damage response cascade involvingMec1, Tel1 and

meiosis-specific components such as Mek1 (Schwacha and Kleckner 1997; Niu et al.

2005;Carballoetal.2008).

The third key characteristic of meiotic HR is a more frequent resolution of joint

molecule intermediatessuchasdHJsor theirprecursors intocrossovers.Mostmeiotic

non-crossover products appear early and result from the processing of unstable

intermediates by SDSA (Allers andLichten2001;Bishop and Zickler 2004). The joint

molecules that can be readily observed are termed single-end invasions (containing

presumably D-loop structures), which later mature into dHJs (Hunter and Kleckner

2001). Most of these joint molecules are resolved synchronously later, which is

triggeredbythephosphorylationofyetundefinedtargetsbytheCdc5kinase(Allersand

Lichten 2001; Sourirajan and Lichten 2008). Although the molecular mechanism

remains unclear, the preferential processing of joint molecule intermediates into

crossoversisachievedbytheusageofameiosis-specificpro-crossoverpathway,which

isresponsibleforupto~80%ofmeioticcrossoversinS.cerevisiae(Zakharyevichetal.

2012). This pathway ensures that every chromosome receives at least one crossover

(positivecrossoverinterference),therebyassuringtheexchangeofgeneticinformation

between the homologous chromosomes. At the same time, the formation of one

crossover suppresses the formation of additional crossovers in its vicinity (negative

crossover interference), making sure that the crossovers are evenly spaced, as two

crossoversinimmediatevicinitylimittheexchangeofgeneticinformationbetweenthe

homologouschromosomes.Thepropernumberof crossoversandtheirdistribution is

thus balanced to enable efficient chromosome segregation and genetic exchange

(Hunter2015).

28

Theexactmechanismofcrossoverinterferenceremainstobedefined.Itisapparent

that it is regulated by the components of the synaptonemal complex that juxtaposes

homologs along their entire length (Sym and Roeder 1994; Hunter 2015). The

interference signaling involves Tel1 (Garcia et al. 2015). Crossover interference also

requirestopoisomeraseII,suggestingthatinterferencemayberegulatedbymechanical

or topological stress along the meiotic chromosome axes (Zhang et al. 2014). The

meioticcrossover-specificpathwayinvolvestheZMMgroupofproteins,whichincludes

Zip1, Zip2, Zip3, Zip4, Spo16,Mer3andMsh4-Msh5 inS.cerevisiae (Lynnet al. 2007;

Shinohara et al. 2008). These proteins form or facilitate the formation of the

synaptonemalcomplex,orfunctionmoredownstreamtostabilizeD-loopstructuresina

waythatprotectstheirdisassemblybymotorproteins(Mazinaetal.2004;Snowdenet

al.2004).Similarlyinmice,theputativeSUMO-conjugatingfactorRNF212wasfoundto

stabilize meiotic recombination factors including MSH4-MSH5 at designated

recombination sites, which is essential for crossing over. This is likely achieved via

sumoylationofselectedtargetsalongthechromosomeaxes,whichfunctionallyinteracts

with HEI10-dependent ubiquitination and proteasomal degradation (Wei et al. 2003;

Reynoldsetal.2013;Raoetal.2017).

Thecrossover-specificresolution inbuddingyeast,plantsandmammalsdepends

onthenucleaseactivityofMlh3,whichisapartoftheMlh1-Mlh3heterodimer(Lipkinet

al.2002;Nishantetal.2008;Zakharyevichetal.2012;Ranjhaetal.2014;Rogachevaet

al.2014).Unexpectedly,Exo1was foundtohaveanon-catalytic function incrossover

resolutiontogetherwithMlh1-Mlh3(Zakharyevichetal.2010).HowtheZMMproteins

and Mlh1-Mlh3 achieve the crossover-specific resolution of dHJs or their precursors

remains to be described (Fig. 7c). Interestingly, only crossovers from the ZMM-Mlh1-

Mlh3pathway– termedClass Icrossovers–display interference. Jointmolecules that

areprocessedbythestructure-specificnucleases(Mus81-Mms4,Slx1-Slx4andYen1in

yeast) result in both crossovers and non-crossovers. Crossovers resulting from these

nucleases are interference independent and account for ~20% of crossovers in S.

cerevisiae(delosSantosetal.2003;Zakharyevichetal.2012).Incontrast,Mus81-Eme1

generates most meiotic crossovers in the budding yeast Schizosaccharomyces pombe,

while the MEI-9, an XPF-like protein, resolves meiotic joint molecules in Drosophila

(Boddyetal.2001;Yildizetal.2002).

7 Role of recombination proteins in promoting the stability of DNA replication

forks

29

In addition to repairingDSBs, recombinationproteins have critical functions in other

pathwaysofDNAmetabolism. Specifically, recombination is oneof thepathways that

contribute to the repair of DNA crosslinks, which has been covered elsewhere (Hinz

2010). Furthermore, recombination in multiple ways promotes the stability of

replicating DNA (Branzei and Szakal 2017). Abnormalities such as protein blocks,

chemicalmodifications,abnormalsecondarystructuresorcollisionsofreplicationforks

withthetranscriptionmachinerycanimpedeDNAreplication.Dependingonthenature

ofthereplicationstress,cellsutilizevariousmechanismsthathelpthemdealwiththese

situations(Fig.10a-d).Thisincludesre-primingofDNAreplicationand/ortranslesion

DNAsynthesis,whichleadtolesionbypass.Postreplicativerepaircanfillanygapsleft

behind by the replication machinery (Bianchi et al. 2013; Garcia-Gomez et al. 2013;

Mouronetal.2013;BranzeiandSzakal2016;GuilliamandDoherty2017;Vaismanand

Woodgate2017).Insomecasesthereplicationforkcanfallapart,resultinginasingle-

endedDSB,which is repairedasdescribed in chapter1.3.Here,wewill focuson two

related functionsofrecombinationproteins thatwereuncoveredonly inrecentyears,

namely in the replication fork reversal and the protection of DNA from nucleolytic

degradationatstalledreplicationforks.

ReplicationforkreversalistheconversionofastalledDNAreplicationforkintoa

four-way junction (Fig. 10b). Fork reversal is achieved by the annealing of the two

nascentDNAstrandsandthere-annealingofthetwoparentalstrands,whichresembles

aDNAdouble-strandbreak.ForkreversalcanthusleadtoDSBformationintheabsence

of templatebreakage(Fig.10b).Althougharisingthroughaverydifferentmechanism,

theresultingfour-wayjunctionisstructurallyidenticaltoaHollidayjunctionthatforms

duringDNArecombination(Fig.10b).Theconceptofreplicationforkreversalhasbeen

proposedalongtimeago(Higginsetal.1976).Inbacteria,ithasbeenestablishedasa

waythatcontributesto therestartofstalledreplication forks. InE.coli, theactionsof

RecA(ahomologueofRAD51), followedbybranchmigrationbyRuvAB,or themotor

activityofRecG,candrivereplicationforkreversal(Seigneuretal.1998;McGlynnand

Lloyd2001;Guptaetal.2014).Foralongtime,thisprocesswasbelievedtorepresent

only a pathological reaction in eukaryotic cells (Sogo et al. 2002; Hu et al. 2012).

Specifically, forkreversal inyeastwasdescribed tooccur incheckpoint-defectivecells

(Lopes et al. 2001). Only recently, it has been demonstrated that replication fork

reversalcanbebeneficialtopreventDNAtemplatebreakageinhumancells,andthatit

isaglobalresponsetoDNAreplicationstress(RayChaudhurietal.2012;Neelsenand

Lopes 2015; Zellweger et al. 2015). Electronmicroscopy revealed that the regressed

30

arms of the replication forks can reach and occasionally exceed 1 kb in length,

implicatingthattheyforminthecourseofanactiveandenzyme-drivenprocess.

One of the first proteins shown to promote replication fork reversal in vivo was

RAD51(Zellwegeretal.2015).Inaccord,RAD51,toalimitedextent,candrivemigration

ofHolliday junctions viapolymerizationonDNA (Rossi et al. 2011).However,RAD51

persehasnomotoractivity,soit isunlikelythatRAD51candriveforkreversalonits

own.ThebestcandidatesforthisfunctionaremotorproteinssuchasDNAhelicasesor

translocases. To this point, the motor protein RAD54 was shown to catalyze fork

reversal invitro in away thatwas stimulatedbyRAD51 (Bugreev et al. 2011).Other

proteins, including SMARCAL1, ZRANB3, HLTF, FBH1, RECQ5 and FANCM were

proposedtofunctioninforkreversalinvitroorinvivo(Kanagarajetal.2006;Garietal.

2008;Betousetal.2012;Cicciaetal.2012;Bhatetal.2015;Fuggeretal.2015;Kileetal.

2015;Kolinjivadietal.2017;Taglialatelaetal.2017;Vujanovicetal.2017).However,

the interplay of these factorswith RAD51 isnotdefined, and it is not clear towhich

extent these motor proteins can function in a redundant manner. Interestingly, fork

reversallikelydoesnotrequireBRCA2,showingthat,unlikeinDSBrepair,thefunction

of RAD51 in promoting fork reversal is BRCA2 independent (Prakash et al. 2015;

Kolinjivadietal.2017;Mijicetal.2017).ForkreversalallowstimeforDNArepairahead

of the fork,ormayresult inDNAdamagebypass(i.e.damage tolerance, incasewhen

onereversedarmtemplatesDNAsynthesisofthecomplementarystrand).Oncethisis

completed,DNAreplicationmustresume.Severalmechanismshavebeendescribedthat

promote the restoration of reversed replication forks, which involve motor proteins

suchasRECQ1,RAD54orthenucleolyticdegradationofthereversedarmsbyMRE11,

EXO1,theDNA2-WRNnuclease-helicasepairorbytheMUS81-dependentcleavage,with

different implication forgenomestability (Bugreevetal.2011;Bertietal.2013;Pepe

andWest 2014a; Thangavel et al. 2015; Lemaconet al. 2017).While replication fork

reversalmaybepathologic insomecases, ithasbeendemonstrated that inhibitionof

forkreversalresults in increasedDNAbreakageandgenome instability,underpinning

theroleofthisprocessinpreventinggenomerearrangements(NeelsenandLopes2015;

Mijicetal.2017).

As described in chapter 3, RAD51 is the main DNA strand exchange protein in

eukaryotes. It remains to be defined whether RAD51 has a structural or a catalytic

functioninreplicationforkreversal.Thereis,however,onekeyfunctionofRAD51that

isDNA repairand recombination independent. Ithasbeendemonstrated thatBRCA1,

BRCA2andRAD51 functiontogether toprotectstalled–andpossiblyreversed–DNA

replication forks from degradation by the MRE11 nuclease (Hashimoto et al. 2010;

31

Schlacheretal.2011;Schlacheretal.2012;Mijicetal.2017).Ithasbeenshownthata

stable RAD51 nucleoprotein filament is required to protect DNA from MRE11

(Kolinjivadietal.2017;Zadorozhnyetal.2017).TheBRCA1andBRCA2 factors likely

act directly or indirectly to stabilize the RAD51 nucleoprotein filament. It is being

discussedwhether the essential function of BRCA2 is to prevent DNA degradation at

stalledforksbystabilizingRAD51,ortopromotecanonicalDSBrepair(RayChaudhuri

et al. 2016; Feng and Jasin 2017). At the same time, unrestricted RAD51 activity at

stalled forks is equally detrimental: the RADX factor was recently identified as a

negative regulator of RAD51 at stalled forks (Dungrawala et al. 2017). These

experiments collectivelydemonstrated that inDNA replication, theRAD51protein, in

addition to performing canonical "recombination" functions has other, previously

unrecognized roles in replication fork reversal and protection from nucleolytic

degradation. To elucidate the underlying molecular mechanisms of these processes

representsachallengetoresearchersinthecomingyears.

Figurelegends

Fig.1AnoverviewofDNAbreaks.aAsingle-strandedDNA(ssDNA)breakariseswhen

only one strand of double-stranded DNA is interrupted. b If an ssDNA break is

encountered by DNA replication, it gives rise to a one-ended double-stranded DNA

(dsDNA) break. c A two-ended dsDNA break forms when dsDNA is broken into two

pieces.

Fig. 2 Anoverviewof the twomainpathways forDNAdouble-strandbreak repair in

human cells.a Main differences between end-joining and homologous recombination

pathways. b DNA double-strand break repair pathway usage gives rise to different

outcomesduringgenomeeditingwithCRISPR-Cas9.Whereasend-joiningoftenresults

inrandommutationsinthevicinityofthebreaksitethatmaydisruptthereadingframe

ofthetargetedgene,homologousrecombinationmaymediatetheprecisereplacement

ofgeneticinformation.

Fig.3AnoverviewofDNAend-joiningrepairmechanisms.aOverviewandmainfactors

of non-homologous end-joining. b Overview and main factors of microhomology-

mediatedend-joining.

32

Fig.4Anoverviewofhomologousrecombinationpathways.Aschematicrepresentation

ofa single-strandannealing,b synthesis-dependentstrandannealing,cbreak-induced

replication and d canonical DNA double-strand break repair pathway that involves

generation of a doubleHolliday junction,which can be processed by either topologic

dissolution(d1)ornucleolyticresolution(d2).Thevariouspathwaysdifferintermsof

mutagenicpotentialandwhethertheyleadtocrossoverornon-crossoverproducts,as

indicated.ThegreentrianglesindicateDNAreplicationsites.NewlysynthesizedDNAis

illustratedusingdashedlines.

Fig. 5 An overview of DNA end resection in human cells. The first (short-range)

resection step involves theMRE11 nuclease and the second (long-range) step either

EXO1orDNA2nuclease.DNA end resection leads to the generationof 3' overhanged

DNAatDNAdouble-strandbreaksites.ThedashedbluelinesindicatedegradedDNA.

Fig. 6 An overview of the RAD51 filament formation (presynaptic phase) and the

invasion of template dsDNA (postsynaptic phase) in human cells. Both positive and

negativeregulatorsoftheprocessareindicated.

Fig.7AnoverviewofdoubleHollidayjunctionprocessingmechanismsinthecanonical

recombinational DSBR pathway. In vegetative cells, double Holliday junctions can be

processedbyeitherdissolution(a)thatinvolveshelicase-coupledtopoisomeraseorby

resolution(b) that involvesnucleolyticcleavageof the junctions.Meioticcells inmost

organismspreferentiallyuseadedicatedcrossoverpathway(c),andtoasmallerdegree

nucleasesthatarecommontobothmeioticandmitoticcells(b).Forsimplicity,human

nomenclature isused.The involvementofnucleases listed inpanelsbandcinhuman

(ormouse)meioticcellsgenerallyremainstobedefined.YeastS.cerevisiaecellsusea

crossover-specific pathway that involves the Exo1-Mlh1-Mlh3 complex and the

structure-specificnucleasesMus81-Mms4,Slx1-Slx4andYen1.Seetextfordetails.

Fig. 8 An overview of specialized roles of the DSB repair pathways in human

lymphocytes.aMechanismofV(D)Jrecombination,whereDNAbreaksare introduced

by RAG1-RAG2. b Mechanism of class switch recombination, where DNA breaks are

indirectly causedby the actionofAID. Inboth cases end-joining, butnothomologous

recombinationpathways,areresponsibleforDNAbreakrepair.

33

Fig. 9 Anoverviewof specialized rolesof thehomologous recombinationpathway in

meiosis. Themaindifferencesbetween recombination inmeiotic andvegetative cells

areindicated.

Fig. 10Anoverviewofpathways thatrelieveDNAreplicationstress inhumancells.a

WhenaDNAreplicationforkencountersanobstacle,itmaybebypassedbytranslesion

synthesis.b A DNA replication fork can also reverse, whichmay temporarily protect

DNAfrombreaking.Replicationforkreversalcanalsoleadtolesionbypass.Factorsthat

may regulate the various steps of replication fork reversal and restart are indicated.

RAD51likelymediatesreplicationforkreversalandprotectsreversedreplicationforks

from degradation by the MRE11 nuclease. c Replication repriming downstream of a

lesion is another mechanism of DNA damage tolerance. d Replication forks can also

break,andbecomeasubstrateforhomologousrecombinationasindicatedinFig.4.

Acknowledgements:Wewould like to thankmembers of the Cejka laboratory (IRB

Bellinzona), Massimo Lopes (University of Zurich) and Kathrin Pieper (Lanzavecchia

laboratory,IRBBellinzona)forcommentsonthemanuscript.Theworkwassupported

bygrantsfromtheEuropeanResearchCouncil(681630)andtheSwissNationalScience

Foundation(31003A_175444)toP.C.Weapologizetocolleagueswhoseworkcouldnot

becitedduetolengthconstraint.

The authors declare no conflict of interest. This article does not contain studies

involvinganimalsorhumanparticipantsbyanyoftheauthors.

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ssDNA breakreplisome

parental stranddaughter strand

parental strand

brokendaughter strand

a

b

cssDNA break two-ended

dsDNA break

one-endeddsDNA break

Figure 1

dsDNA break

mutations

DNA template

• Fast • Slow• Template independent • Requires template• Often mutagenic • Largely accurate• Cell cycle independent • Only S/G2 phase• Only two-ended breaks • Both one and two-ended breaks• No repair of protein blocked ends • OK for protein blocked ends

Cas9

STOP

DNA break repair

End-joining Homologous recombination (HR)

DNA template

Random mutagenesis, frameshiftleads to premature termination

Precise editing, replacement with desired DNA sequence

a

b

End-joining: Non-homologousend-joining (NHEJ) or Microhomologymediated end-joining (MMEJ)

Homologous recombination (HR)

Figure 2

mutations

guide RNA

target dsDNA

Overview of end-joining DNA double-strand break repair pathways

Non-homologousend-joining (NHEJ)

Microhomology-mediatedend-joining (MMEJ)

microhomology at ends (2-20 nt)

two-endeddsDNA break

Dna2, MRN, CtIP, ???

Dna2, MRN, CtIP, ???

Polθ

DNA Ligase III, ???

DNA overhangcleavage

DNA synthesis DNA synthesis

DNA ligation DNA ligation

DNA end protection

limited DNA end processing

no or short (< 4 nt) microhomology at ends

DNA annealing

Ku70-Ku80

XRCC4-XLF-DNA Ligase IV

ArtemisPolλ or Polμ

DNA-PKcsin case of nonligatable DNA ends:

DNA-PKcs

Figure 3

a b

oror

or

dsDNA break

Extended DNA end resection

Annealing at homologous(>20 nt) loci, overhang cleavage

DNA synthesis

Endpoint: non-crossover,deletion at break site

Single-strand annealing (SSA)

Synthesis-dependent strand annealing (SDSA)

Break-induced replication (BIR)

Topological processing of dHJ (Dissolution)

Nucleolytic cleavage of dHJ (Resolution)

Overview of homologous recombination pathways

DNA synthesis

Endpoint: non-crossover

Disruption of D-loop

Invasion of homologous DNA

Second end captureDNA synthesis

DNA synthesis, ligation

D-loop

3’ ssDNA overhang

double Holliday junction (dHJ)

Migration of bothjunctions towardseach other

Endpoint: non-crossover

Endpoint: non-reciprocalcrossover

DNA synthesis viabubble migration

... Later in cell cycle

Cleavage by structure-selectivenucleases along horizontal orvertical lines (random)

Endpoint: reciprocal crossover

Endpoint: non-crossover

Cano

nica

l DN

A d

oubl

e-st

rand

bre

ak re

pair

path

way

via

dou

ble

Hol

liday

junc

tion

template DNA

a

b

c

d1

d2

Figure 4

dsDNA breakOverview of DNA end resection pathways

short 3’ ssDNA overhang

DNA adduct

MRE11RAD50

NBS1

Recruitment of MRN complex

CtIP

Recruitment of phosphorylated CtIP

P

Activation of MRE11endonuclease

EXO1WRN or BLMDNA2

5’3’

3’3’

or

Shor

t ran

ge re

sect

ion

long 3’ ssDNA overhanglong 3’ ssDNA overhang

5’

Resection backtoward endMRE11 exonuclease

1st step:Short-range DNA end resection

2nd step:Long-range DNA end resection

Figure 5

RPA

RAD51BRCA2

RAD51 �lament

remodelled RAD51 �lament

D-looptemplate DNA

DNA synthesis

RAD54

Resected RPA-coated3’ ssDNA overhang

3’

BRCA2 exchanges RPA forRAD51 on overhanged ssDNA,blocks dsDNA binding of RAD51

RAD51 nucleprotein �lament

Active RAD51 �lament

Invasion of template dsDNA, RPA stabilizes D-loop by binding to displaced ssDNA

Removal of RAD51 from dsDNA by RAD54, DNA synthesis by Polδ or Polε

RAD51 paralogsRAD54RAD52PALB2

RAD51 paralogs

BRCA1

RAD51 paralogsDSS1SWS1 ?

RECQ5FBH1PARIBLM

RTEL1BLMRECQ1RAD54

RPA

Pres

ynap

tic

phas

ePo

stsy

napt

ic p

hase

Positiveregulation:

Negativeregulation:

RAD54

Figure 6

Convergent branch migration (dissolution)

Nucleolytic resolution

double Holliday junction (dHJ)

Hemicatenane

Endpoint: non-crossover

Convergently migrated dHJ

Nucleolytic resolution interference-dependent

Endpoint: Crossover

Endpoint: non-crossover

OR

Endpoint: Crossover

BLM-TopoIIIα-RMI1-RMI2

SLX1-SLX4 MUS81-EME1(XPF-ERCC1) SM

XSM

GEN1

MLH1-MLH3 and EXO1, ZMM proteins, ?

Mechanism:Helicase & topoisomerase

Helicase & topoisomerase

Topoisomerase

Mechanism: Nuclease

Mechanism: Nuclease

Vegetative cells Meiotic cells

a

b

c

Figure 7

Recognition of RS12/23 by RAG1-RAG2

NHEJ-mediated DSB repair

Vn Vn + 1 Jn Jn + 1 Jn + 2 Jn + 3

DNA cleavage by RAG1-RAG2

Lost

RS23 sequenceRS12 sequence

hairpin endblunt end

properly joined V and J segments

MRNArtemis

Ku70-Ku80DNA-PKcs

XRCC4-XLF, DNA ligase IV

circular DNA

V(D)J Cμ Cδ Cγ Cε

V(D)J Cμ Cδ Cγ Cε

UG

GU

GG

G

G

GG

G

G

IgM

V(D)J

Cμ Cδ

Cγ Cε

EXO1

Mismatch repair

Base excision repair

DNA double-strand break

+

+

Lost

V(D)J Cγ Cε

IgG

NHEJ or MMEJ-mediated DSB repair

RAG1-RAG2RAG1-RAG2

Vn Vn + 1 Jn Jn + 1 Jn + 2 Jn + 3

Loop formation, alignment of RS12 and RS23 sequences

a V(D)J recombination b Class switch recombination

MRN

AID

T cellsB cells

AID

Figure 8

Cα

Cα

Cα

Cα

Diploid cell

Haploid gametes

dsDNA break formation

Homologous recombination

Crossover formation

1 meioticdivision

2 meioticdivision

Meiotic vs. Vegetative cells

DNA double-strand breaks:SPO11-induced, programmed Accidental

Preferential repair template:Homologous chromosome Sister chromatid

Genetic outcome:At least one crossover perchromosome, evenly spaced

Non-crossovers >> crossovers

Maintenance of genome stability Proper chromosome seggregationExchange of genetic information

st

nd

Strand exchange protein(s):RAD51, DMC1 RAD51

FUNCTION:

Figure 9

replisome

parental strand

lagging strand

STOPlesion

leading strand

parental strand

lagging strand

STOP

lesion

leading strand

RAD51, RAD54 SMARCAL1ZRANB3FBH1, RECQ5FANCM, ?

RAD51 �lament

MRE11PTIP?

replisome

parental strand

lagging strand

leading strandRECQ1

MUS81-EME1/EME2RAD54RAD51 ?

replisome

parental strand

lagging strand

leading strand

Swith back to replicative polymerase

translesion polymerase

Postreplicative gap repair

Lesion ahead of replication fork

ContinuedDNA replication

RADX

BRCA2

a Translesion synthesis

b Replicationfork reversal

c Replicationrepriming

d Break inducedreplication

Replication fork restoration

Fork breakageMUS81-EME1RECQ5

See Figure 4

lesion

lesion

Figure 10

STOP

STOP