TIGRTIGRTIGRTIGR

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Topics of Discussion

• DNA Repair

• Why study evolution of repair?

• Evolution of specific pathways with examples from recent genome projects (e.g., A. thaliana, Vibrio cholerae, Shewanella putrefaciens, Buchnera aphidicolum symbiont)

• Big picture – evolutionary origins of repair

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Damage is not just to DNA

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General Mechanisms of Resistance to Cellular Damaging Agents

• Damage protection/prevention

• Damage tolerance

• Repair and recovery

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Classes of DNA Repair

• Direct repair– Photoreactivation– Alkylation transfer– DNA ligation/non-homologous end joining

• Excision repair– Base excision repair– Mismatch excision repair– Nucleotide excision repair

• Recombinational repair

TIGRTIGRTIGRTIGR

Endonuclease

NUCLEOTIDE and

MISMATCH EXCISION

BASE EXCISION

Exonuclease, Helicase, Polymerase

Damage Recognition

* *

N-glycosylase

AP endo

Ligase

Exonuclease, Polymerase

Excision Repair Outline

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Generation of single-strand overhang

Initiation, alignment

RecA RecFOR

Rad52

Strand invasion

DNA synthesis

Branch migration and resolution

RuvABC RecG,RUS?

Rad54?

RecA Rad51,55,57

RecBCD RecE,T RecQ,J

Rad50, MRE11, XRS2

Recombination Outline

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“Nothing in biology makes senseexcept in the light of evolution.”

T. H. Dobzhansky (1973)

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Why Study Evolution and Repair?

• Repair variation leads to differences in evolutionary patterns within and between species.

• Evolutionary analysis can identify mutation/repair biases.

• Evolutionary studies can improve our understanding of repair proteins and pathways.

• Comparisons of repair genes can be used to infer evolutionary history.

• Information on mutation processes improves sequence and phylogenetic analysis.

• Evolutionary analysis is required to infer the origins and history of repair processes.

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Steps in Phylogenomic Analysis

• Create database of genes of interest

• Presence/absence of homologs in complete genomes

• Phylogenetic trees of each gene family

• Infer evolutionary events (gene origin, duplication, loss and transfer)

• Refine presence/absence (orthologs, paralogs, subfamilies)

• Functional predictions and functional evolution

• Analysis of pathways

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Pathway Biochemical Activity(s). |-------------------------------------Bacteria------------------------------------| |-----Archaea------| |--Eukarya---|Protein Name(s)

Bacter ial NERUvrA Binds damaged DNA + + + + + + + + + + + + + + + - - - - -UvrB Helicase, 3' incision endonuclease + + + + + + + + + + + + + + + - - - - -UvrC 5' incision endonuclease + + + + + + + + + + + + + + + - - - - -UvrD Excision helicase + + + + + ++ + + + ++ + + + + ++ - - - + +MFD Transcription repair coupling + + + + + + - - + + + + - + - - - - - -

Eukaryotic NER

RecognitionRad14 (XPA) Binds damaged DNA - - - - - - - - - - - - - - - - - - + + +RFA1/RPA1 ssDNA binding w/ RFA2,3 - - - - - - - - - - - - - - ± - - - + + +RFA2/RPA2 ssDNA binding w/ RFA1,3 - - - - - - - - - - - - - - - - - - + ++ +RFA3/RPA3-human ssDNA binding w/ RFA1,2 - - - - - - - - - - - - - - - - - - - + +RFA3/RPA3-yeast ssDNA binding w/ RFA1,2 - - - - - - - - - - - - - - - - - - + +

InitiationRad3 (XPD) (ERCC2) TFIIH component – helicase - - - - - - - - - - - - - - - - - ± + + +Rad25 (XPB) (ERCC3) TFIIH component – helicase - - - - - - - - - + - + - + - - + + + + +SSL1 (p44) TFIIH component - - - - - - - - - - - - - - - - - - + + +TFB1 (p62) TFIIH component - - - - - - - - - - - - - - - - - - + + +TFB2 (p52) TFIIH component - - - - - - - - - - - - - - - - - - + + +TFB3 (MAT1) TFIIH component - - - - - - - - - - - - - - - - - - + + +TFB4 (p34) TFIIH component - - - - - - - - - - - - - - - - - - + + +CCL1 (CyclinH) TFIIH component - - - - - - - - - - - - - - - - - - + + +Kin28 (CDK7) TFIIH component - protein kinase - - - - - - - - - - - - - - - - - - + + +

IncisionRad2 (XPG) (ERCC5) 3' incision (flap endonuclease) - - - - - - - - - - - - - - + + + + + + +Rad10 (ERCC1) 5' incision endonuclease w/ Rad1 - - - - - - - - - - - - - - - - - - + + +Rad1 (XPF) (ERCC4) 5' incision endonuclease w/ Rad10 - - - - - - - - - - - - - + + + + + + +

SpecificityRad4 (XPC) Repair of inactive DNA - - - - - - - - - - - - - - - - - - + + +Rad23 (HHRAD23) Repair of inactive DNA - - - - - - - - - - - - - - - - - - + ++ +Rad7 Repair of inactive DNA - - - - - - - - - - - - - - - - - - + +Rad16 Repair of inactive DNA - - - - - - - - - - - - - - - - - - + + +Rad26 (CSB) (ERCC6) Transcription-repair coupling - - - - - - - - - - - - - - - - - - + + +CSA (ERCC8) Transcription-repair coupling - - - - - - - - - - - - - - - - - - ± + +

Nucleotide Excision Repair

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Pathway Biochemical Activity(s). |-------------------------------------Bacteria------------------------------------| |-----Archaea------| |--Eukarya---|Protein Name(s)

Initiation

RecBCD pathwayRecB ExoV Helicase + + + - - - - - - + + - - + - - - - - -RecC ExoV Nuclease + + + - - - - - - + ±+ - - + - - - - - -RecD ExoV Helicase + + + - ± ± - - - + ±+ - - + - - - - - -

RecF pathwayRecF Assists RecA fi lamentation + + - - + + - - + + - + - + - - ± - ± ±RecJ 5'-3' ssDNA exonuclease + + + + + + - - + - + + + + - - - - - -RecO Binds ssDNA, assists RecF? + + + - + + - - + + - - - + - - - - - -RecR ATP binding, assists RecF? + + + ±+ + + - - + + - + + + - - - - - -RecN ATP binding + + + + + + - - + + - + + + - - ± - - -RecQ 3'-5' DNA helicase + + + - ± + - - + - - + - + - - - - + ++ +

RecE pathwayRecE/ExoVIII 5'-3' dsDNA exonuclease + - - - - - - - - - - - - + - - - - - -RecT Binds ssDNA, promotes pairing + - - - + + - - - - - - - + - - - - - -

SbcBCD pathwaySbcB/ExoI 3'-5' ssDNA exonuclease + + - - - - - - - - - - - + - - - - - -SbcC dsDNA exonuclease (w/ sbcD) + - - - ±+ + - - + - + + + + ± ± ± ± ± ± ±SbcD dsDNA exonuclease (w/ sbcC) + - - - - + - - + - + + + + ± ± ± ± ± ± ±

AddAB PathwayAddA/RexA Exonuclease + helicase w/ AddB - - + - + + - - - - - + - + - - - - - -AddB/RexB Exonuclease + helicase w/ AddA - - + - + + - - - - - - - + - - - - - -

Rad52 pathwayRad52, Rad59 n/a - - - - - - - - - - - - - - - - - - ++ + +Mre11/Rad32 Nuclease w/ Rad50 ± - - - ± ± - - ± - ± ± ± ± + + + + + + +Rad50 Nuclease w/ Mre11 ± - - - ± ± - - ± - ± ± ± ± + + + + + + +

RecombinaseRecA, Rad51 DNA binding, strand exchange + + + + + + + + + + + + + + + + + + ++ ++ ++

Branch migration/resolution

Branch migrationRuvA Binds junctions. Helicase w/ RuvB + + + + + + + + + + + + - + - - - - - -RuvB 5'-3' junction helicase w/ RuvA + + + + + + + + + + + + - + - - - - - -

RecG Resolvase, 3'-5' junction helicase + + + + + + - - + + + + + + - - - - - -

ResolvasesRuvC Junction endonuclease + + + + - - - - + + - + - + - - - - - -RecG Resolvase, 3'-5' junction helicase + + + + + + - - + + + + + + - - - - - -Rus Junction endonuclease + - - - - - - - - ±+ - - ±+ + - - - - - -CCE1 Junction endonuclease - - - - - - - - - - - - - - - - - - + +

Other recombination proteinsRad54 n/a - - - - - - - - - - - - - - - - - - + + +Rad55 n/a - - - - - - - - - - - - - - - - - - + + +Rad57 n/a - - - - - - - - - - - - - - - - - - + + +Xrs2 Assists Rad50/MRE11? - - - - - - - - - - - - - - - - - - + +

Recombinational Repair

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Evolution of Specific Pathways

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Photoreactivation and Photolyases

• All photoreactivation is carried out by enzymes in the photolyase family

• Two main classes of photolyases – class I and class II – are distantly related to each other and likely the result of an ancient duplication

• PhrI and PhrII missing from most species for which complete genomes are available.

• Many cases of functional change (e.g., CPD -> 6-4) and some are not even involved in DNA repair

• Many of the eukaryotic proteins appear to be of an organellar ancestry

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Uses of Evolution : Photoreactivation• All known enzymes that perform photoreactivation are part of

a single large photolyase gene family

• Some members of the family do not function as photolyases, but instead work as blue-light receptors

• If a species does not encode a member of the photolyase gene family, it likely does not have photoreactivation capability

• If a species encodes a photolyase, one cannot conclude it has photolyase activity

• Position of photolyase homologs within photolyase tree helps predict what activities they have

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Phr.S thyp

PHR E. coli

ORFA00965*********

phr.neucr

Phr.Tricho

Phr.Yeast

Phr.B firm

phr.strpy

phr.haloba

PHR STRGR

pCRY1.huma

phr.mouse

phr2.human

phr2.mouse

phr.drosop

phr3.Synsp

ORF02295.Vibch********

phr.neigo

ORF01792.Vibch*******

Phr.Adiant

Phr2.Adian

Phr3.Adian

phr.tomato

CRY1 ARATH

phr.phycom

CRY2 ARATH

PHH1.arath

PHR1 SINAL

phr.chlamy

PHR ANANI

phr.Synsp

PHR SYNY3

phr.Theth

Rh.caps

MTHF type Class I CPD Photolyases

6-4 Photolyases

Blue Light

Receptors

8-HDF type CPD

Photolyases

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Photolyases in A. thalianaphr.chlamycry2.tomatPHH1.CRY2.PHR1 SINALCry3.AdianCRY1/hy4.Aphr.BrevibPhr.Cordi.ORF05094.CPhr.RhocaPhr.BacfiPhr.EntfaPhr.StrpyPhr.Pseae.Phr.Yerpe.Phr.EcoliPhr.S thypPhr.SaltyPhr.Shepu.A00965.VibPhr.YeastPhr.NeucrPhr.TrichoPhr.SYNY3 Phr.AnaniPhr.Synsp Phr.ThethPhr.Mycav.Phr.MycsmCT12574.FlARATH3 MSJPhr.flypCRY1.humaPhrL.MousePhrL2.humaPhrL2.Mous295.VibchPhr2.SynspARATH2 T30ARATH5 F6A1792.VibchPhr.NeimePhr.NeigoPhr.HalhaPhr.Strgr

Crys Group with -Proteobacteria

Other Bacteria

Eukaryotic

Cyano/Plastid

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Alkyltransferases

• All known alkyltransferases are members of a single gene family

• Found in most but not all species

• Likely present in LUCA

• Ada protein in E. coli originated by fusion between an alkyltransferase and a transcription-regulatory domain

• Gram-positive bacteria have the Ada domain fused to an alkylation glycosylase instead of alkyltransferase

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AlkA Domain (O6-Me-G glycosylase)

Ogt Domain (O6-Me-G alkyltransferase)

Ada Domain (transcriptions regulator)

Ada E. coli

Ada H. infl

Ogt E. coli

Ogt H. infl

Ogt Gram+

Ogt D. radio

AlkA Gram+

AlkA E. coli

MGMT Euks

Alkylation Repair Genes

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DNA Ligases

• Two major ligase families

• Ligase I– NAD dependent– Found in all bacteria and only in bacteria

• Ligase II– ATP dependent– Found in all Archaea and eukaryotes– Found in some bacteria– Duplicated in many eukaryotes

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DNA Ligases in A. thaliana

ARATH1 F4N21.14YEAST-GP-600039YEAST-SW-DNLI YEASTYEAST-GP-3515ARATH1 F13F21.31ARATH1 T23G18.1ARATH1 T6D22.10CELEG C29A12.3DROMECG560AERPE-gi|5104764.AQUAE-gi|2983805DROME-CG17227ARATH5 MUL3 11YEAST-SW-DNL4 YEASTDROMECG12176ARCFU-gi|2648829METJA-gi|1590924METTH-gi|2622703ARCFU-gi|2649996PYRHO-gi|3258051PYRFUPf 1527421

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Mismatch Excision Repair• Core of process highly homologous between bacteria and eukaryotes (all use

MutS and MutL homologs).

• Eukaryotes encode multiple MutS and MutL homologs, not all of which are involved in mismatch repair.

• Two major MutS groups– MutS-I proteins involved in MMR and MutS-II proteins involved in chromosome segregation.

• MutS1 and MutL missing from many bacteria, especially pathogens. Other MMR proteins also defective in some.

• Few homologs in Archaea – some encode MutS2, none encode MutS1, and some may encode MutL.

• Some evolutionary and functional relationships to restriction-modification systems (MutH, MED1, Vsr).

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95

79

MSH6

MSH3

MutS1

MutS-I Mismatch Repair MSH1

MSH2

100

100

74

MutS-II Chromosome Crossover & Segregation

MSH5

MSH4

MutS2

96

100

95

100

100

90

96

25

55

85

60

Proposed duplication

61/89

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5

1

2

3

4

E. coli

H. influenzae

N. gonorrhoeae

H. pylori

Syn. sp

B. subtilis

S. pyogenes

M. pneumoniae

M. genitalium

A. aeolicus

D. radiodurans

T. pallidum

B.burgdorferi

A. aeolicus

S pyogenes

B. subtilis

Syn. sp

D. radiodurans

B. burgdorferi

Syn. sp

B. subtilis

S. pyogenes

A. aeolicus

D. radiodurans

B. burgdorferi

MutS2

MutS1

A. B.

Gene Duplication

Gene Duplication

Ancient Duplication in MutS Family

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Parallel Loss of MutLS

Lost in mycoplasmal lineage (present in B. subtilis and S. pyogenes)

Lost in M. tuberculosis lineage (found in some other highGC Gram-positives)

Lost in H. pylori / C. jejuni lineage (present in many other Proteobacteria)

Possibly lost in Euryarchaeota lineageDefective in many “wild” E. coli and S. typhimurium strains

Loss of genes may give an advantage in some conditions by increasing mutation rate or recombination rate between species.

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Nucleotide Excision Repair• Bacterial and eukaryotic systems are not-homologous, despite having

very similar mechanisms

• Most of the eukaryotic and bacterial proteins originated within each of these domains

• Some of the eukaryotic proteins are shared with Archaea (Rad1, Rad2, Rad25).

• All free-living bacteria encode UvrABCD. B. aphidicolum encodes Mfd but not UvrABCD.

• UvrABC also found in one Archaea.

• Some functional and evolutionary relationships with drug resistance and transport

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Evolution of UvrA Family

UvrA2UvrA2 S. coelicolor

DrrC S. peuceteus

UvrA2 D. radiodurans

Duplication in UvrA family

UvrA1

UvrA H. influenzae

UvrA E. coli

UvrA N. gonorrhoaea

UvrA R. prowazekii

UvrA S. mutans

UvrA S. pyogenes

UvrA S. pneumoniae

UvrA B. subtilis

UvrA M. luteus

UvrA M. tuberculosis

UvrA M. hermoautotrophicum

UvrA H. pylori

UvrA C. jejuni

UvrA P. gingivalis

UvrA C. tepidum

uvra1 D. radiodurans

UvrA T. thermophilus

UvrA T. pallidum

UvrA B. burgdorefi

UvrA T. maritima

UvrA A. aeolicus

UvrA Synechocystis sp.

UvrA1

UvrA2

OppDF

UUP

NodI

LivF

XylG

NrtDC

PstB

MDR

HlyB

TAP1

CFTR, SUR

A. ABC Transporters B. UvrA Subfamily

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UvrA Evolution

Diversification of ABC family

UvrA

UvrAC UvrAN

UvrA1C UvrA1N UvrA2C UvrA2N

ABC1ABC2

ABC

Tandem Duplication

Gene Duplication

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Base Excision Repair Glycosylases

• Distribution patterns highly uneven but some glycosylases have been found in all species

• Some are ancient enzymes, probably presence in LUCA (e.g., MutY-Nth), others more recent (e.g., TagI).

• Many families are distantly related to each other (e.g., Ogg, AlkA, MutY-Nth)

• Many cases of gene duplication, loss and possibly transfer, especially from organellar genomes to nucleus

• Orthologs frequently have different specificity

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A. thaliana TAG homologsC. crescentus

A. thaliana_ 5 K23L20 1

A. thaliana_ 3 MBK21.7

A. thaliana_ 1 F23A5.15

A. thaliana_ 1 T24D18.7

A. thaliana_5 MTI20 23

A. thaliana_1 F9E10.6

V. cholerae

H. influenzae

E.coli

M. tuberculosis

N. meningitidis A

N. meningitidis B

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AP Endonucleases

• All species encode either Nfo or Xth homologs. Some encode both.

• Only Nfo: mycoplasmas, Aquifex, M. jannascii, yeast

• Only Xth: many bacteria, A. fulgidus, humans (so far)

• Both: E. coli, B. subtilis, M. tuberculosis, M. thermoautotrophicum

• Both Nfo and Xth are likely ancient.

• Many cases of gene loss of one or the other, but never both

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Recombinational Repair• RecA homologs found in all free-living species (B. aphidicolum encodes

RecBCD but not RecA)

• Most recombination initiation pathways are of recent origin– RecBCD, RecE within Proteobacteria/Gram-positives– RecF within bacteria– AddAB within low-GC gram-Positives– SbcCD may be of ancient origin (possibly homologous to MRE11/Rad50)

• Resolution pathways also somewhat recent origin– CCE1 within eukaryotes– RuvABC, RecG near origin of bacteria– Rus within bacteria (phage origin?)

• Many cases of gene loss in initiation, resolution pathways.

TIGRTIGRTIGRTIGR1Myx.xanth

2Myx.xanth

MBBAD17TF******

0.1

Xen.bovieXen.nemat

Pr.vulgariPr.mirabilEnt.agglo

Y.pestisS.marcesce

E.coliShig.flexShig.sonn

Shepu.tigVib.angui

Vib.cholerPs.oleovor

Ps.marginaPs.fluores

Ps.putidPs.aerugiPs.aePAM

Az.vinelan

Ac.calcoacAc.sp.ADP

Past.haemH.influenz

Past.multoActinobaci

Aer.salmonXa.oryza

Xa.citriXa.campes

B.pertussiPs.cepaciChrom.vino

Mthmon.claMthphy.met

Mthbac.flaNitrosomon

L.pneumopNe.gonorr

Ne.meningiT.ferrooxiRhb.phaseRh.leguminA.tumefaciRh.melilot

Br.abortusBlastochlo

Rhps.paluAceto.polAceto.alt

Gluc.oxydAq.magnet

Zym.mobiliCaul.cresc

Prcs.denitRho.sphae

Rho.capsu

He.pyloriHe.pylori2

Cmp.jejuniCmp.fetus

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RumB R391

A05970

ImpB

MucB

UmuCs

RulB

RumB******

DinP1DinP2

UvrX

DinP3

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Big Picture: Evolutionary Origin

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Likely Ancient Repair Processes/Proteins

Process Proteins

Mismatch repair MutL, MutSAP endonuclease Xth, NfoRecombinase RecA/RadA/Rad51Alkylation reversal Ogt/MGMTPhotolyase PhrII, PhrIdGTP/GTP clean up MutTBase excision glycosylases MutY/Nth, AlkA, Ung?Recombination endonuclease SbcC/Rad50, SbcD/MRE11Other SMS, Lon, UmuC

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Originated within Bacteria

Process Proteins

Mismatch repair MutH, VsrAlkylation reversal Ada (fusion of Ogt)Base excision Fpg-Nei, TagIRecombination initiation RecFJNOR, AddAB,

RecBCD, RecET, SbcBRecombination resolution RecG, RuvABC, RusNucleotide excision repair UvrABCDTranscription-coupled repair MFDInduction LexAOther SSB, LigaseI

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Originated within EukaryotesProcess Proteins

Mismatch repair duplications of MutS, mutLBase excision 3MG?Recombinase duplication of RecARecombination initiation duplications of RecQRecombination resolution CCE1Nucleotide excision repair Most XPs, TFIIH, etc.Transcription-coupled repair CSA, CSBInduction P53Non-homologous end joining XRCC4, Kus, DNA-PKcsOther RFAs, Rad52-59, XRS2

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Originated in Eukaryote-Archaea Lineage

Process Proteins

Base excision Ogg

Nucleotide excision repair Rad1, Rad2, Rad25?

Ligation LigaseII

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Ambiguous Origin

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Repair Genes in Archaea

• All species: RecA,MRE11, Rad50, MutY-Nth, Ogt, Rad2, Lig-II, PCNA

• UvrABCD in M. thermoautotrophicum• PhrI and PhrII in some species• Variety of glycosylases in some species• No Ung homologs in any species, but

alternative glycosylases have Ung activity• Rad1 in many species.• New Holliday junction resolvase

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DNA Repair Genes in D. radiodurans Complete Genome

Process Genes in D. radiodurans

Nucleotide Excision Repair UvrABCD, UvrA2Base Excision Repair AlkA, Ung, Ung2, GT, MutM, MutY-Nths,

MPGAP Endonuclease XthMismatch Excision Repair MutS, MutLRecombination Initiation Recombinase Migration and resolution

RecFJNRQ, SbcCD, RecDRecARuvABC, RecG

Replication PolA, PolC, PolX, phage PolLigation DnlJdNTP pools, cleanup MutTs, RRaseOther LexA, RadA, HepA, UVDE, MutS2

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Problem:

List of DNA repair gene homologs in D. radiodurans genome is not significantly different from other

bacterial genomes of the similar size

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Unusual Features of D. radiodurans DNA Repair Genes

Process Genes

Nucleotide excision repair Two UvrAs

Base excision repair Four MutY-Nths

Recombination RecD but not RecBC

Replication Four Pol genes

dNTP pools Many MutTs, two RRases

Other UVDE

TIGRTIGRTIGRTIGR

-Ogt-RecFRQN-RuvC-Dut-SMS

-PhrI-AlkA-Nfo-Vsr-SbcCD-LexA-UmuC

-PhrI-PhrII-AlkA-Fpg-Nfo-MutLS-RecFORQ-SbcCD-LexA-UmuC-TagI

-PhrI-Ogt-AlkA-Xth-MutLS-RecFJORQN-Mfd-SbcCD-RecG-Dut-PriA-LexA-SMS-MutT

-PhrI-PhrII?-AlkA-Fpg-Nfo-RecO-LexA-UmuC

-PhrI-Ung?-MutLS-RecQ?-Dut-UmuC

-PhrII-Ogg

-Ogt-AlkA-TagI-Nfo-Rec-SbcCD-LexA

-Ogt-AlkA-Nfo-RecQ-SbcD?-Lon-LexA

-AlkA-Xth-Rad25?

-AlkA-Rad25

-Nfo

-Ogt-Ung-Nfo-Dut-Lon

-Ung

-PhrII

-PhrI

Ecoli

Haein

Neig

o

Help

y

Bacsu

Str

py

Mycg

e

Mycp

n

Borb

u

Tre

pa

Syn

sp

Metj

n

Arc

fu

Mett

h

Hu

man

Yeast

BACTERIA ARCHAEA EUKARYOTES

from mitochondria

+Ada+MutH+SbcB

dPhr

+TagI?+Fpg

+UvrABCD+Mfd

+RecFJNOR+RuvABC

+RecG+LigI

+LexA+SSB

+PriA+Dut?

+Rus+UmuD

+Nei?+RecE

tRecT?

+Vsr+RecBCD?

+RFAs+TFIIH

+Rad4,10,14,16,23,26+CSA

+Rad52,53,54+DNA-PK, Ku

dSNF2dMutSdMutLdRecA

+Rad1+Rad2

+Rad25?+Ogg+LigII

+Ung?+SSB,

+Dut?

+PhrI, PhrII+Ogt

+Ung, AlkA, MutY-Nth+AlkA

+Xth, Nfo?+MutLS?

+SbcCD+RecA

+UmuC+MutT

+LondMutSI/MutSII

dRecA/SMSdPhrI/PhrII

+Sprt3MG

+Rad7+CCE1

+P53dRecQdRad23+MAG?

-PhrII-RuvC

tRad25

+TagI?

+RecT

tUvrABCD

tTagI ?

Gain and Loss of Repair Genes

TIGRTIGRTIGRTIGR

TIGRTIGRTIGRTIGR

Repair Studies in Different Species(determined by Medline searches as of 1998)

Humans 7028E. coli 3926S. cerevisiae 988Drosophila 387B. subtilits 284S. pombe 116Xenopus 56C. elegans 25A. thaliana 20Methanogens 16Haloferax 5Giardia 0

TIGRTIGRTIGRTIGR

Evolution of Repair Summary• Mycoplasmas have lost many repair genes which may

explain high mutation rate.• Mismatch repair genes absent in many pathogens (is high

mutation rate advantageous?)• Whole pathways frequently lost as units (e.g., MutLS).• May be able to predict pathway interactions by correlated

loss of genes.• Archaeal genomes have few homologs of bacterial or

eukaryotic repair proteins.• Some eukaryotic repair proteins have likely mitochondrial

and plastid ancestry• Many ancient duplications (MutS, SNF2, UvrC).• Some unusual distributions (XPB, UvrABCD)

TIGRTIGRTIGRTIGR

TIGRTIGRTIGRTIGR

AcknowledgementsAcknowledgementsNIEHS•Ben Van Houten

TIGR•Craig Venter•Claire Fraser•John Heidelberg•Owen White•Steve Salzberg

Stanford•Phil Hanawalt•Rick Myers•D. Crowley

Louisiana State University•John Battista

U.C. Berkeley•Michael Eisen•A. J. Clark

Funding•DOE, OBER•NIH•NSF

Other•J. Laval•F. Taddei•A. Britt•J. Miller

TIGRTIGRTIGRTIGR

TIGRTIGRTIGRTIGR

Unusual Distributions

• XP-B like gene in some bacteria and some Archaea.• LigaseII in M. tuberculosis, B. subtilis, and A. aeolicus• UvrABCD in M. thermoatuotrophicum• Mycoplasmas and some low GC gram positives do not have

any Holliday junction resolving homologs (RuvC, RecG, Rus)

• Mycoplasmas are the only species without MutY-Nth homologs

• MutS2 unevenly distributed among bacteria, Archaea• Genes in RecF pathway not always present as a unit• Uracil glycosylase missing from Archaea and some bacteria

TIGRTIGRTIGRTIGR

Big Picture: Duplication and Loss

TIGRTIGRTIGRTIGR

Genes Lost in Mycoplasmal Lineage

Process Protein

Base excision repair MutY/Nth, AlkARecombination initiation RecF pathway, SbcCDRecombination resolution RecG, RuvCMismatch repair MutLSTranscription coupled repair MFDInduction LexADirect repair PhrI, OgtAP endonuclease XthOther MutT, Dut, PriA, SMS

TIGRTIGRTIGRTIGR

Parallel Loss of MutLS

Lost in mycoplasmal lineage (present in B. subtilis and S. pyogenes)

Lost in M. tuberculosis lineage (found in some other highGC Gram-positives)

Lost in H. pylori lineage (present in many other Proteobacteria)Possibly lost in Euryarchaeota lineageDefective in many “wild” E. coli and S. typhimurium strains

Loss of genes may give an advantage in some conditions by increasing mutation rate or recombination rate between species.

TIGRTIGRTIGRTIGR

Need for Experimental Studies in Archaea

• No novel repair genes cloned in Archaea. All repair genes show homology to repair genes in other species.

• Many novel repair genes found in bacteria and eukaryotes because of experimental work in these species.

• Since novel repair pathways appear to evolve frequently in bacteria and eukaryotes, there is a need for more genetic and experimental studies of repair in Archaea.

TIGRTIGRTIGRTIGR

Repair Genes in all Archaea

Process Protein

Nucleotide excision repair Rad2, Rad1 ±

Recombination RecA, Mre11, Rad50

Replication PolB, PCNA

Ligase Ligase II

Base excision repair MutY-Nth

dNTP pools MutT family

Alkyltransferase Ogt in all species

TIGRTIGRTIGRTIGR

DNA Repair Gene Summary

• Most of the standard eukaryotic DNA repair genes are found

• Some likely plastid repair genes are found

• Some duplications relative to other species

TIGRTIGRTIGRTIGR

Acknowledgements

• Genome duplications: S. Salzberg, J. Heidelberg, O. White, A. Stoltzfus, J. Peterson

• Genome sequences and analysis: J. Heidelberg, T. Read, H. Tettelin, K. Nelson, J. Peterson, R. Fleischmann, D. Bryant

• Horizontal transfers: K. Nelson, W. F. Doolittle

• TIGR: C. Fraser, J. Venter, M-I. Benito, S. Kaul, Seqcore

• $$$: DOE, NSF, NIH, ONR

TIGRTIGRTIGRTIGR

Evolution of Uracil Glycosylase

• Many non-homologous proteins have uracil-DNA glycosylase activity (Ung, GPADH, MUG, cyclin)

• Therefore, absence of homologs of these genes should not be used to infer likely absence of activity

• However, presence of homologs of Ung and MUG genes can be used to indicate presence of activity because all homologs of these genes have this activity

TIGRTIGRTIGRTIGR

Ambiguous Origin

Process Proteins

Base excision 3MG, GT MMR, Ung

Nucleotide excision repair Rad25

Recombination initiation RecQ

Other Dut

TIGRTIGRTIGRTIGR

Big Picture: Distribution Patterns

TIGRTIGRTIGRTIGR

Present in All Bacteria

Process Proteins

Nucleotide Excision Repair

Recombinase

Replication PolA,C

Single-strand DNA Binding SSB

Ligase LigaseI

TIGRTIGRTIGRTIGR

Present in All Free-Living Bacteria

Process Proteins

Nucleotide Excision Repair UvrABCD

Recombinase RecA

Replication PolA,C

Single-strand DNA Binding SSB

Ligase LigaseI

TIGRTIGRTIGRTIGR

Present in Most Bacteria

Process Protein

Nucleotide excision repair UvrABCDHolliday junction resolution RuvABCRecombination RecA; RecJ, RecGReplication PolA,C; PriA; SSBLigase DnlJTranscription-coupled repair MfdBase excision repair Ung, MutY-NthAP endonuclease Xth

TIGRTIGRTIGRTIGR

Present in Bacteria or Eukaryotes(But Not Both)

Process Bacteria Eukaryotes

Transcription-coupled repair CSB, CSA

Mismatch strand recognition MutH -

Nucleotide excision repair UvrABC XPs, TFIIH, etc.

Recombination initiation RecBCD, RecF KU, DNA-PK

Holliday junction resolutionRuvABC CCE1

Base excision -

Inducible responses LexA P53

TIGRTIGRTIGRTIGR

Evolution of Alkyltransferases

• All known alkyltransferases share a conserved, homologous alkyltransferase domain

• Therefore, if a species does not encode any protein with this domain, it likely does not have alkyltransferase activity

• If a species does encode an member of this gene family, it likely has alkyltransferase activity

TIGRTIGRTIGRTIGR

Standard Eukaryotic Repair GenesPathway Genes

Mismatch Repair MSH2-6, MutLs

Base Excision Repair Ogg, MutY-Nth, Tag, 3MG, Ung

Nucleotide Excision Repair

XPA, Rad1, Rad2, Rad3, Rad10, Rad25, etc

Recombination MRE11, Rad50 Rad51

Direct Repair Phr, Dnl1

Other PCNA, Dut, Lon

TIGRTIGRTIGRTIGR

Missing Eukaryotic Repair Genes?

Pathway GenesMismatch RepairBase Excision RepairNucleotide ExcisionRepair

XPA, Btf2, Btf3,Kin28

RecombinationDirect Repair OgtOther

TIGRTIGRTIGRTIGR

MSH6.PombeMSH6.YeastGTBP.Arath IV.At4g02070ARATH T10M13.8ARATH AGAA.3MSH7.ArathGTBP.MouseGTBP.HumanY47G6A.11.CelegansMSH3.HumanREP1.MouseMSH3.Arath IV M7J2.90SWI4.pombeMSH3.yeastMUTS BORBUMUTS TREPAMutS.Cloac.blastMutS.Clodi.blastMutS.SynspMutS.Chlte.blastMutS.Porgi.blastMutS.TheaqMutS.Theaq caldMutS.ThethMutS.ThemaMutS.EcoliMutS.SaltyMutS.Yerpe.blastMutS.VibchMutS.HaeinMutS.Actin.blastMutS.Actin.blastMutS.Pasmu.blastMutS.Shepu.blastMutS.Neime.TIGRMutS.Neigo.blastMutS.AzoviMutS.Pseae.blastMutS.Thife.blastMutS.Entfa.blastMutS.Strmu.blastMutS.Strpy.blastHexA.StrpnMutS.Staau.blastMutS.BacsuMutS.RicprMUTS RHIMEMutS.Caucr.prelimMutS.AqupyMutS.AquaeMutS.Chltr.?MutS.ChlpnMSH2.RatMSH2.HumanMSH2.MouseMSH2.XenlaMSH2.NeucrMSH2.YeastMSH2.Arath3Mus1.MaizeMSH2.PombeSPE1.DromeH26D21.2.CelegMSH1.SpombeMSH1.YeastMSH1.Canal.blastMSH4.CelegMSH4.YeastMSH4.CanalMSH4.humanARATH3 MQC12.24MSH5.YeastMSH5.HumanMsh5.MouseMSH5.CelegmMutS.Saco.glaucum.Muts2.MetthMutS2.PyrhoMutS2.PyrabMutS2.Helpy.TIGRMutS2.Helpy99MutS2.CamjeMutS2.Deira.TIGRMutS2.ThemaMutS2.BacsuMutS2.StaauMuts2.Entfa.blastMuts2.Strmu.blastMutS2.Strpy.BlastMutS2.Strpn.blastMuts2.Cloac.blastMuts2.Clodi.blastMutS2.SynspARATH 5 MJP23 7Muts2.Chlte.blastMutS2.BorbuMutS2.AquaeMuts2.Porgi.blastMutS2.Arath 1 F16G16.7

89

8654

7233

15

93

3855

3010035

93100

27

3882100

3112

17

20

96

12

35

4830

26

33

66

100 100

834310082

8260100

9766100

100

61

10015

9798 77100

78

54100

24

100100

89

85887240

3810093 79

100

92

88

80

51 76

67

4653100

55

100 100

100100

4950 179

27 39872317

584510077

9526

70

Bootstrap

MutS2

MSH5

MSH4

MSH1

MSH2

MutS1

MSH3

MSH6

TIGRTIGRTIGRTIGR

E.coliShig.flexneriShig.sonnei

Ent.agglomeransY.pestisS.marcescensXen.bovienii.editXen.nematophilus

Pr.vulgarisPr.mirabilisShepu.tigr

Vib.anguillarumVib.cholerae%MPLAAer.salmonicidaActinobacillus actinomycetemcoH.influenzaePast.multocidaPast.haemolyticaPs.oleovorans

Ps.marginalisPs.fluorescensPs.putida

Ps.aeruginosaPs.aePAM7Az.vinelandiiAc.calcoaceticusAc.sp.ADP1

Mthmon.claraMthphy.methylotrophusMthbac.flagellatumNitrosomonas.blastChrom.vinosum

L.pneumophilaT.ferrooxidansXa.oryzaeXa.campestrisXa.citri

B.pertussisPs.cepacia

Ne.gonorrhoeaNe.meningitis.TIGR

Blastochloris.viridisRhps.palustrisRhb.phaseoliRh.leguminosarumA.tumefaciensRh.melilotiBr.abortus

Aq.magnetotacticum.editAceto.polyoxogenesAceto.altoacetigenesGluc.oxydansZym.mobilisPrcs.denitrificansRho.sphaeroidesRho.capsulatusCaul.crescentusRic.prowazekii

reca.wolbachia.blastHe.pyloriHe.pylori2

Cmp.jejuniCmp.fetus1Myx.xanthus2Myx.xanthusblast.geobacterblast.desulf

Tmtg.maritimaTrep.pallidumTr.denticolum.blastBor.borgdorferi

Lept.biflexaLept.interrogansSpir.platensisSyn.7942Ana.variabilis

Syn.7002Syn.6803A.thaliana

A.thaliana.chr3A.thaliana.chr2

Ureo urolyticumMycp.genitaliumMycp.pneumoniaePhytoplasma sp

Mycp.mycoidesMycp.pulmonas

De.radioduransThe.aquaticusThe.thermophilus

Chlfx.aurantiacusDehalo.blastChl.trachomatisChl.pneumoniae

Cory.glutamicumCory.pseudotuberculosis

Strpm.coelicolorStrpm.lividansStrpm.ambofaciensStrpm.violaceusStrpm.rimosusMycb.tuberculosisMycb.bovis.sanger.fr2Mycb.avium.blastMycb.lepraeMycb.smegmatisAmycolatopsis mediterraneiBifido.breve

Chb.tepidumPorp.gingivalisBact.fragilisPrev.ruminocolaList.monocytogenesB.subtilisBac anthracis.blastEnt faecalis.blastStaph.aureus

Strc.pneumoniaeStrc.parasanguisStrc.pyogenes1Lct.lactisAch.laidlawiireca.blast.carboxyClost.perfringensCl.acetybutylicum.blast

Aq.pyrophilusAq.aeolicus

100

18

4

3

4

25

10

30

46

25

25

59

66

49

43

87

71

61

100

81

4587

55

55100

100

96

100

23

83100 75

100

93

100

96100

100

7

8

306590100

35

100

67

99

100

40

4936

44

54

96

85

87997885

94

93100

98

84

18

56100100

82

32

8095

17

449993

96

99

9768

39

94

10

21

2351

88

100100

68

97

100

9

48

60

100

9652

100

57

100

935299

99

993656100

39

10078

30

77

70

76

7364 60

100

10088

84

100

Bootstrap

SpirochetesCyanobacteria

MycoplasmasD/TGreen Non-SulfurChlamydia

High GC Gram +

Green Sulfur

Low GC Gram +

Hydrogenobacteria

TIGRTIGRTIGRTIGR

DDM1.Arath VYFK8.yeast

F24P17 ArathIIISNF2L2 yeast

SNF2L1 yeastSNF2L Celeg

ISW1 flySNF2LB human

SNF2L humanhSNF2h humanSNF2L Plasmodium

BRG1 humanBRG1 mouse

BRG1 chickenBRM human

BRM chickenBRM fly

C52B9 CelegF01G4 Celeg

STH1 yeastSNF2 yeastSPAC1250 pombe

Brm PlasmodiumT3B23.4 ArathII

T3F17.33.ArathIIT04D1.4 Celeg

human.6016932Mi2 human

CHD3 humanT14G8 CelegF26F12 Celeg

CHD3 flypickle.Arath II

F13D4.130 ArathIIT19K24.8 ArathV

CHD1 yeastSPAC3G6 Spombe

CHD1 SpombeCHD1 humanCHD1 mouse

CHD1Z chickenCHD2 human

CHD1 fly?H06O01 Celeg

F14O4.6.Arath IIT14P1.22.ArathII

YGP0 YEASTKIAA1259.human

CT11902.flyF2809.150.Arath IIIYDR334w.yeast

SPAC11E3 SpombeSRCAP.Human

CT27348.flyCT27330.fly

ETL.mouseKIAA1122.human

CG5899.flyM03C11.CelegSPAC20G8.Spombe

SPAC25A8.SpombeFUN30.yeast

F5O4.14.Arath IICSB.human

Rad26.yeastRhp26.Spombe

MSF3.14.Arath IIF11M21.32.Arath IF53H4 Celeg

T25N20.13.Arath IF16F14.11.Arath II

F3K23.21.Arath IIRad54L1.celeg

ATRX.humanATRX.mouse

CG4548.flyC27B7.Celegnas

F22O13.8.Arath IRad54.flyRad54.chicken

Rad54.mouseRad54.human

Rad54.NcrassaRhp54.pombe

RAD54.yeastRAD54B.Human

Rad54L1.yeastRad54L1.SpombeAC22F3

Mot1.arath

MOT1.yeastMot1.fly

TAFII170.humanF15D4.Celeg.3880600

Drp1CG10445

CG10445.flyLODE.DrosophilaT23H2.Celeg

F54E12.CelegHuf2.Human.3702846

GTA.Autographa virusGTA.O pseudo virus

GTA.BudwormHIP116A.human

RUSH1a.RabbitTNF.MouseRAD5.yeast

RAD8.PombeRIS1.yeast

AC17A2.pombeT19D16.2.Arath

F11P17 ArathIC19G10.02.pombe

RAD16.yeastRhp16.Spombe

YLR247c.YeastCT22559.CG7376SPAC144.Spombe

T7D17.5.Arath IIiridi virus

HepA.AfuHepA.Pyrho.3257311

seq.pyrfu.ncbiBTM.TM0990

HepA.Deira.ORFB00056HepA.HalYqhH.Bsu

HepA.DnoHepA.Eco

HepA.Vibch.03168hepa.shewa

HepA.HinF11A17 Arath I

humanmouseHarp.Bos taurus

C16A3.CelegansCG3753.fly

GVC.ORF02241HepA2.Chltr

BTC.ORF00253BCP.ORF00856

HepA2.MgeHepA2.Mpn

snf2.StrcoHepA2.BsuHepA2.Syn

HelZ.Mtub.2909552RadB.Sulso

HepA2.Dra.TIGR.partHepA.Chltr.3329163

BTC.ORF00041BCP.ORF00420

HepA2.Spy.partHepA2.Bce

79

65

34

42

55

36

40

22

30

35

100

40

45

43

75

22

16

25

100

97

53

96

97

98

100100

50

100

100

100

100

54100

100

100

10086

39

60

100

86

77

42

100

62

100

100

100

99

10090

9393

75

100100

97

90

10038

53

100

69

100100

69

80

10071

100

10080

54

49

47100

8593

45

49

100100

100

60100

58100

100

96

100

100100

98

10092

65

100

82100

100

24

68

46

51

95

79

64100

64100

100100

28

38

10082

98

49

4673

100

34100

9959

60

60

100

31

30100

100

25

39

35

10094

54

100

68

10076

70

100100

100

75

44

5465

100100

Bootstrap

DDM1

SNF2L

SNF2

CHD3

CHD1

YGPO

SRCAP

ETL1

CSB

ARTX

RAD54

MOT1

RAD16

HEPA1

HARP

HEPA2

TIGRTIGRTIGRTIGR

Homo sapiensAGMonkeyMonkeyMacacaRabbitWoodchuckMouseRatMastomysM auratusC griseusBos taurusBos.indicusBos.primigeniusOvis ariesFelis catusp53.guineapigChickenXenopuszebrafishp53.barbelp53.catfishTroutp53.Xiph maculatusp53.Xiph maculatusp53.Xiph helleriOryziasp53.tetraodonFlounderSquidp53.flycusp.humanp73h.mousep51a.humanp51d.humanp63a.humanp63b.mousep63g.humanp63g.mousep63a.mouseket.ratp73.barbelp73#3.humanp53#2.humanp73#1.human

99

100

52

83

10053

85

6187 90

9760

99

95

100

53

93100

55100

71

73

94

100

74

100

Bootstrap

TIGRTIGRTIGRTIGR

Mitochondrial Repair Genes

Pathway Genes Mismatch Repair Base Excision Repair TagI Nucleotide Excision Repair

Recombination RecA? Direct Repair Phr Other Lon, SSB, AlkB

TIGRTIGRTIGRTIGR

Plastid Repair Genes

Pathway Genes Mismatch Repair MutS2a, MutS2b Base Excision Repair Fpg Nucleotide Excision Repair

Mfd

Recombination RecA, RecG, SMS Direct Repair Phr Other Lon, PolI, SSB?