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Progress in Biophysics and Molecular Biology 117 (2015) 182e193

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Progress in Biophysics and Molecular Biology

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Envisioning the dynamics and flexibility of Mre11-Rad50-Nbs1complex to decipher its roles in DNA replication and repair

Julien Lafrance-Vanasse a, Gareth J. Williams a, John A. Tainer a, b, *

a Life Science Division, 1 Cyclotron Road, Berkeley, CA 94720, USAb The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

a r t i c l e i n f o

Article history:Available online 7 January 2015

Keywords:Mre11-Rad50-Nbs1CtIPAllosteryDouble strand break repairDynamicsConformational change

Abbreviations: AFM, atomic force microscopy;mutated; ATR, ataxia telangiectasia and Rad3-relatedminus domain; DSB, DNA double strand break; DSBREM, electron microscopy; FHA, fork head-associatedrecombination repair; MRN, Mre11-Rad50-Nbs1; Njoining; SAXS, small-angle X-ray scattering.* Corresponding author. Life Science Division, 1 C

94720 USA.E-mail address: jatainer@lbl.gov (J.A. Tainer).

http://dx.doi.org/10.1016/j.pbiomolbio.2014.12.0040079-6107/Published by Elsevier Ltd. This is an open

a b s t r a c t

The Mre11-Rad50-Nbs1 (MRN) complex is a dynamic macromolecular machine that acts in the first stepsof DNA double strand break repair, and each of its components has intrinsic dynamics and flexibilityproperties that are directly linked with their functions. As a result, deciphering the functional structuralbiology of the MRN complex is driving novel and integrated technologies to define the dynamic struc-tural biology of protein machinery interacting with DNA. Rad50 promotes dramatic long-range allosterythrough its coiled-coil and zinc-hook domains. Its ATPase activity drives dynamic transitions betweenmonomeric and dimeric forms that can be modulated with mutants modifying the ATPase rate to controlend joining versus resection activities. The biological functions of Mre11's dual endo- and exonucleaseactivities in repair pathway choice were enigmatic until recently, when they were unveiled by thedevelopment of specific nuclease inhibitors. Mre11 dimer flexibility, which may be regulated in cells tocontrol MRN function, suggests new inhibitor design strategies for cancer intervention. Nbs1 has FHAand BRCT domains to bind multiple interaction partners that further regulate MRN. One of them, CtIP,modulates the Mre11 excision activity for homologous recombination repair. Overall, these combinedproperties suggest novel therapeutic strategies. Furthermore, they collectively help to explain how MRNregulates DNA repair pathway choice with implications for improving the design and analysis of cancerclinical trials that employ DNA damaging agents or target the DNA damage response.

Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1832. Functional architecture of the MRN complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1833. A minimal model integrating diverse MRN functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1844. Long-range allostery: the Rad50 coiled-coil and zinc hook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1855. Functional dynamics of the Mre11-Rad50 ATPase head complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1866. Mre11 conformations act in Rad50's ATP-dependent dimerization and DNA sculpting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1887. Nbs1 and CtIP regulate pathway choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1898. Clinical and therapeutic opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1899. Combining biophysics and molecular biology to understand MRN's structural flexibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

10. Perspective and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

ATM, ataxia telangiectasiaprotein; BRCT, BRCA1 C-ter-, double strand break repair;domain; HRR, homologousHEJ, non-homologous end-

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J. Lafrance-Vanasse et al. / Progress in Biophysics and Molecular Biology 117 (2015) 182e193 183

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

1. Introduction

DNA double strand break repair (DSBR) is a fascinating processthat involves a plethora of proteins in a complex choreography ofdynamic events in which the Mre11-Rad50-Nbs1 complex (MRN)plays critical roles. Double strand breaks (DSB) are created byexposure to ionizing radiation or chemicals, and by endogenouscellular events, including DNA replication and V(D)J recombination.For more than 40 years, scientists have strived to determine howDSBRworks. By combining results piece by piece and by developingnewmethods, they are revealing the integrated mechanisms of thiscrucial phenomenon. We know there are many other interactingcomponents for homologous recombination such as those thatpromote homologous DNA pairing in meiotic recombination (Zhaoet al., 2014); however here we focus on the MRN complex, whichacts in the first stages of DSBR.MRN has emerged as one of themainprotein complexes at multiple stages in the process, impactingpathways for homologous recombination repair (HRR) and bothclassical and alternative non-homologous end-joining (NHEJ)(Hammel et al., 2011; Hopfner et al., 2002b; Shin et al., 2004; G. J.Williams et al., 2014, 2010; Zha et al., 2009). It detects DSBs, tethersthe ends of the broken chromosomes, activates DNA damageresponse pathways and nucleolytically processes the DNA ends. Afascinating characteristic of MRN is that these diverse functions arepossible due to its flexibility and dynamic properties that are finallybeing unveiled.

In 2002 an article in Nature revealed multiple conformations ofthe MR complex by electron microscopy (EM) (Hopfner et al.,2002a) and in 2005 a second Nature article presented a videocaptured by atomic force microscopy (AFM) of the MRN complexinteracting with DNA (Moreno-Herrero et al., 2005; Williams andTainer, 2005), allowing the implications of the dynamics and flex-ibility to be examined in the context of Mre11 and Rad50 crystalstructures. These papers opened the door for the scientific com-munity to visualize MRN in its dynamic splendor. In the solutionAFM video, the long coiled-coils of Rad50 are moving between twodifferent states upon DNA binding. In their DNA-bound form, thetwo 500 Å-long coiled-coils of an MRN dimer seem to be straightand rigid. When the complex leaves the DNA, the coiled-coils adopta relaxed, bent position, similar to what is seen by EM (de Jageret al., 2001). These observations revealed that the complex ishighly dynamic over a large spatial area.

In the last 10 years, tremendous efforts have been done to betterunderstand the dynamic nature of MRN, its structure and itscellular functions. Indeed, MRN has diverse and critical functions:DSB detection, DNA end tethering, ATM activation, DNA end pro-cessing and replication fork protection or resection (reviewed in(Paull and Deshpande, 2014; Schiller et al., 2014; Symington, 2014;Williams et al., 2010)). The emerging picture is that MRN usesflexible and dynamic structures to integrate its ATPase and nucleaseenzymatic activities with proteineprotein and protein-DNA in-teractions to control biological outcomes at a DSB. To study dy-namic Mre11-Rad50 complexes, we and other researchers in thefield have used the power of optimal model systems includinghyperthermophiles (reviewed in Shin et al., 2014). Furthermore,multiple and combined technologies have been employed, whichprompted the design of our synchrotron beamline to combinecrystallography and X-ray scattering (Classen et al., 2013). Indeed,

the MRN complex has provided problem-driven development ofmultiple biophysical metrics and methods, which is noted alongwith the biological results.

In this review we cover the most recent conceptual advancesabout MRN in the context of foundational results. We first explorethe current models on its roles in different pathways. Then weexamine how the flexibility and dynamics of MRN's componentsand partners are linked with their function. Rad50 is capable oflong-range allostery and its activities are controlled by its ATPasefunctions, which impacts the nucleolytic activities of Mre11. Mre11sculpts dsDNA ends and ssDNA differently and inhibitors weretherefore able to separately block either endonuclease or exonu-clease activities by blocking Mre11-mediated DNA conformationalchanges. Also, Nbs1 and CtIP are responsible for important dynamicproteineprotein interactions with the Nbs1 C-terminus helping toconnect MRN to ATM activation and signaling. Finally, there isgrowing evidence that MRN can be targeted for cancer therapeuticsand knowing its dynamics andmolecular mechanism is thus crucialfor developing new therapeutic strategies. Overall, we aim to helpthe reader appreciate the dynamic and structural aspects of MRNfunctions. This knowledge includes growing evidence that struc-tural dynamics can regulate repair pathway selectivity and controlbiological outcomes. This appreciation of the MRN complex hasgeneral biological implications, especially for the Rad50 ABCATPase superfamily (Hopfner and Tainer, 2003), for structure-specific nucleases (Tsutakawa et al., 2014), and for phosphory-lated protein binding by FHA and BRCT domains (Reinhardt andYaffe, 2013).

2. Functional architecture of the MRN complex

The structure of the whole MRN complex or of its individualcomponents has been examined by multiple techniques: X-raycrystallography, small-angle X-ray scattering (SAXS), analytical ul-tracentrifugation, inductively coupled plasma mass spectrometry(ICP-MS), dynamic light scattering (DLS), atomic force microscopy(AFM) and electron microscopy (EM). Initially, proteins fromarchaea and bacteria were used, since they are more stable in theirpurified form, but recently eukaryotic Mre11 and Nbs1 have alsobeen studied, strengthening the previous structural findings. Beforegoing into further details with the MRN complex, it is useful toconsider a structural overview of each of its components (Fig. 1).

Rad50 is a member of the ABC ATPase superfamily and hasprovided insights of broad interest across the superfamily (Hopfnerand Tainer, 2003). It is the largest protein of theMRN complex, with1312 amino acids in the human enzyme (Fig. 3). Both the N- and C-terminal ends of the protein form the “head” ABC-ATPase domain,which contains the Walker A/B motifs and signature motif char-acteristic of this family of ATPases (Moncalian et al., 2004). It hasboth ATPase and adenylate kinase activities in vitro (Bhaskara et al.,2007; Paull and Gellert, 1999), but its major biological activityseems to be as an ATPase. Between the two termini of the protein,anti-parallel coiled-coil domains extend the length of the proteinup to ~500 Å (Hopfner et al., 2000). Opposite to the head and at thetip of the coiled-coil, a CXXC motif creates a zinc-hook that facili-tates dimerization of Rad50 (Cahill and Carney, 2007; Hopfneret al., 2002a). Indeed, two zinc-hooks (a total of four cysteines)can be attached together by coordinating a Zn2þ atom. But this is

Fig. 2. Model of MRN's functions in DSB repair. (A) MRN first detects and interactswith the DNA end. (B) If the process goes into HRR, MRN moves away from the break(shownwith an arrow) and recruits CtIP. (C) CtIP induces changes in MRN leading to anopen complex capable of nuclease activities. (D) After an initial endonuclease cut, MRNresects from 3ʹ to 5ʹ towards the DSB, with its exonuclease activity, while EXO1/BLM orDNA2/BLM resects from 5ʹ to 3ʹ. (E) In NHEJ or HRR, the DNA tethering functions ofMRN can be used. Color-coding is the same as in Fig. 1.

Fig. 1. Architecture of the Mre11-Rad50-Nbs1 complex with its partner CtIP. The Rad50dimer is in orange and yellow (for each monomer) and the coiled-coils are representedas helices extending towards the zinc-hook. The Mre11 dimer is shown in blue anddark blue and interacts with Nbs1, shown in green (different shades represent thedifferent domains). Nbs1 interacts with CtIP through its FHA domain (dark green).Interaction with ATM is shown by a black arrow. The dotted lines represent disorderedprotein linkers.

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not the only part of Rad50 that interacts with another subunit. Thehead also dimerizes upon ATP binding: the N-terminus of oneRad50 interacts with the C-terminus of the other (and vice-versa),to form two complete active sites (Hopfner et al., 2000).

Like Rad50, Mre11 (708 residues in the human enzyme) is adimer and it dimerizes through its core domain, located at the N-terminus of the sequence (Fig. 5). This core phosphodiesterasedomain has single stranded DNA (ssDNA) endonuclease and 30e50

double stranded DNA (dsDNA) exonuclease activities (Hopfneret al., 2001). In the active site, five histidines, two aspartic acidsand an asparagine coordinate two Mn2þ ions and are important forcatalysis (Hopfner et al., 2001). Attached to the core domain, a“capping” domain is thought to help discriminate between ssDNAand dsDNA substrates and to be involved in orientating the DNAsubstrate within the active site (Das et al., 2010; R. S. Williams et al.,2008). The C-terminal domain of Mre11 is predicted to be disor-dered or flexible, except a small region forming a-helices thatinteract with the base of Rad50's coiled-coils (Lammens et al., 2011;Williams et al., 2011). The rest of the C-terminal domain isresponsible for proteineprotein and protein-DNA interactions andis subject to post-translational modifications, including argininemethylation (Boisvert et al., 2005; D�ery et al., 2008). The functionalcomplex formed by the Mre11 and Rad50 dimers is conservedthroughout all the three domains of life (named MR or M2R2) (Limet al., 2011; M€ockel et al., 2012), revealing this as a critical complexfor cell biology.

While absent in bacteria and archaea, eukaryotes have Nbs1(Xrs2 in Saccharomyces cerevisae), a phosphoprotein-binding andadapter subunit that plays an important role in connecting M2R2 toother DSB repair and signaling molecules. Although the stoichi-ometry is not fully known, recent structures support a model inwhich two Nbs1 interact with M2R2, as opposed to only one(Schiller et al., 2012). Thus it is a “M2R2N2” hetero-hexamericcomplex that is often implied when referring to the MRN com-plex. Starting from the N-terminus, Nbs1 possesses a fork head-associated (FHA) domain and two breast cancer-associated 1C ter-minus (BRCT) domains known to bind phosphoproteins (Fig. 5).

These three domains are responsible for interactions with severalphosphorylated proteins (see Section 7), like CtIP (R. S. Williamset al., 2009). On the other hand, the C-terminal half of Nbs1 isflexible, with the C-terminus containing adjacent motifs thatinteract with Mre11 and ATM.

3. A minimal model integrating diverse MRN functions

MRN is one of the first proteins to detect the DNA ends createdat a DSB. Yet, how the whole complex precisely interacts with DNAin vivo is not known. After this initial DNA binding event, MRNinteracts and activates ATM kinase to initiate DSB signaling andparticipates in DSB repair processes. In this section we present anoverview of MRN's mechanism in the two major pathways, HRRand NHEJ.

In HRR, the MRN complex has to move 100-200 base pairs awayfrom the end of the DNA (Fig. 2AeD) (Shibata et al., 2014). Then, theprotein CtIP (Sae2 in yeast) stimulates the endonuclease activity ofMre11, which makes a cut in one strand of the DNA (Cannavo andCejka, 2014). It is currently debated whether or not CtIP has anintrinsic endonuclease activity (Makharashvili et al., 2014; H. Wanget al., 2014a). From that point, Mre11 30e50 exonuclease activityresects the DNA strand back towards the break (Cannavo and Cejka,2014; Garcia et al., 2011; Shibata et al., 2014; Zakharyevich et al.,2010). Simultaneously, the helicase/nuclease resection machinery(BLM in complex with either Exo1 and/or DNA2) carries out long-range 50e30 resection from the endonuclease cut in the oppositedirection (Gravel et al., 2008; Mimitou and Symington, 2008; Zhuet al., 2008). These combined activities create the >1000 bpssDNA overhang necessary for HRR. We know that the Rad50coiled-coils are necessary in this process, but how they act preciselyis an important question that remains to be solved. One model forthe function of the Rad50 coiled-coils in both NHEJ and HRR is totether the two DNA ends together. Instead of interacting within thedimer, the zinc-hooks interact with another MRN-DNA complex toattach them together (Fig. 2E). In yeast and in human cells, Rad50and its hook keeps a DNA double-strand break from becoming achromosome break (Hohl et al., 2011; Lobachev et al., 2004;

Fig. 3. Long-range allostery is a crucial feature of Rad50. Top: ATP binding induces rotation of the C-lobe relative to the N-lobe of Rad50's head domain. This is communicated to thezinc-hook domain through the coiled-coils, possibly by helix sliding. Bottom: schematic of Rad50's sequence with arrows depicting segments of flexibility and dynamics.

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Wiltzius et al., 2005). We are starting to understand how all theseprocesses need to be controlled and finely tuned. This is what weexplore in greater details in the next sections.

4. Long-range allostery: the Rad50 coiled-coil and zinc hook

The 500 Å-long coiled-coil of Rad50, joining at one end the ABC-ATPase head and at the other end the zinc-hook, is its most strikingfeature. The length of the coiled-coil seems to be important forRad50's function, since shortening it has dramatic effects in vivo,impairing telomere maintenance and meiotic DSB formation (Hohlet al., 2011). Furthermore, NHEJ is severely affected by shorteningthe coiled-coils. On the other hand, homologous recombinationfunctions are stable with a shorter coiled-coil (~300 amino acidsremoved), but not a very short coiled-coil (~500 amino acidsremoved) (Hohl et al., 2011). This could be an effect of a loss offlexibility in the coiled-coils (Hohl et al., 2011).

Indeed, EM and AFM images show that the coiled-coil is flexibleand adopts multiple conformations (de Jager et al., 2001, 2004;Hopfner et al., 2001; Moreno-Herrero et al., 2005). These imagesshow that as homodimers, their zinc-hooks and/or their ATPaseheads attach two Rad50s. In some cases, the two coiled-coils are inproximity throughout their length to form a straight and rigid rod,as seen by AFM and EM (Anderson et al., 2001; Hopfner et al.,2002a, 2001). But most of the time, when both ends are attached,the coiled-coils have substantial flexibility (van Noort et al., 2003),and appear to create a curved, ovoid loop. Despite the structuralinsights, the importance and function of the various conformationsof the Rad50 coiled-coils are still poorly understood.

How the structure of the zinc-hook relates to its function is alsopuzzling. Its crystal structure shows that it can join two coiled-coilsin almost opposite directions (Hopfner et al., 2002a). At the apex ofthe coiled-coils, a loop of twelve amino acids curves back and formsthe hook. In the middle, the cysteines of a CXXC motif coordinate aZn2þ ion. The two pairs of cysteines form a tetragonal geometryaround the Zn2þ ion. This structural model agrees with EM imagesshowing arch-shaped Rad50 coils from single dimers (Hopfneret al., 2002a). Furthermore the configuration seen in the crystalstructure also agrees with a model where a Rad50 dimer wouldattach to another Rad50 dimer, to bridge two DNA ends (Fig. 2E).

Mutational analysis of this motif and surrounding residuesprovided insight into its function. If the cysteines are mutated inyeast, survival after ionizing radiation is compromised and theMre11-Rad50 interaction is impaired (Hopfner et al., 2002a), but

the Rad50 dimerization through the head, the Mre11-Rad50interaction or the interaction with DNA are not compromised(Cahill and Carney, 2007). Furthermore, removing the zinc-hook inyeast also removes the DSB repair functions of MRN and impairstelomere maintenance, but does not affect Rad50's capability toassociate with chromatin (Wiltzius et al., 2005). On the other hand,mutating the zinc-hook cysteines in mammalian cells does affectthe recruitment and stability of MRN to chromosomal DSBs. Thismutant fails to activate ATM, is defective in end resection and ATRactivation, and thus is defective in initiating HRR-mediated DSBrepair (He et al., 2012). This is further confirmed by hookmutants inmouse (Roset et al., 2014). Since Rad50 cysteine mutants are em-bryonic lethal, as is the Rad50 knock-out, it was necessary tomutate residues around the cysteines in order to obtain a milderphenotype. Thus two mutants were created in mouse (Rad5046:S679R þ P682E and Rad5047: V683R). While homozygotes are notviable, heterozygotes can survive and are relatively proficient inDNA repair. Yet, they have problems in DNA damage responsesignaling through the ATM pathway, which greatly enhancestumorigenesis. At the molecular level, those Rad50-hook mutantsstill have the ability to dimerize and bind Zn2þ, but are engaging inheterotypic dimerization.

These pieces of evidence inform us that Rad50 engages in long-range allostery through the coiled-coils: from the zinc-hook to theATPase domain and vice versa (Fig. 3). The coiled-coils are not justa spacer or a rod to improve the reach of Rad50's interactions, butthere is a mechanical link between both ends of Rad50. The zinchook functions are associated with NHEJ, which is consistent withits function in bridging DNA molecules that are interacting withthe head. In that pathway, the zinc-hook interacts in an inter-molecular fashion. On the other hand, a long coiled-coil is notnecessary in homologous recombination, but the zinc hook is stillimportant for intramolecular interactions. We emit the idea thatthe hook can undergo a structural transition to orient the coiled-coils of an MRN dimer in different orientations relative to oneanother. This transition would be communicated from the ATPasedomain, through the coiled-coils. Interestingly, a similar mecha-nism is seen in dyneins, which are protein-motors involved inintracellular transport and other processes. Briefly, the dyneinATPase head is connected to a microtubule binding domain (MBD)by a 130 Å-long anti-parallel coiled-coil. The current “helix sliding”model suggests that conformational changes driven by ATP hy-drolysis cause the two helices of the coiled-coil to slide, whichchanges the orientation and affinity of the MBD towards tubulin

Fig. 4. Rad50 ATP binding and hydrolysis controls its conformational state and pathway choice (A) Crystal structures showing the Mre11-Rad50 head without nucleotide (top, PDB3QG5) and bound to nucleotide (bottom, PDB 3AV0). Mre11 (surface representation) and Rad50 (cartoon representation) are colored by subdomains as indicated (the color of thefont matches the color of the domain). In the nucleotide-free state, Rad50 monomers are flexible and conformationally sample the closed state, as indicated by dashed-arrows. (B)Top: Crystal structures of P. furiosus Rad50 without nucleotide (PDB 3QKS) show a core cavity (green surface) adjacent to the signature motif (magenta) that remodels upon ATP-binding. The L802W mutation (PDB 4NCH) partially fills the core cavity, destabilizing the ATP-bound closed state. In contrast, the R805E variant (PDB 4NCI) destabilizes the Rad50monomer resulting in a stabilization of the ATP-bound closed state. Bottom: The effects of the mutations compared to wildtype Rad50 on Mre11-Rad50 conformational states andactivities are depicted.

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(Carter et al., 2008; Kon et al., 2009; Redwine et al., 2012). Thisreleases the dynein from tubulin and allows movement. ForRad50, changes in the ATPase domain could also cause the coiled-coils to slide and transmit information to the zinc hook via long-range allostery. To support this model, it was seen with AFMthat a transition in the orientation of the coiled-coils is dependenton DNA binding by the MRN head (Moreno-Herrero et al., 2005).Furthermore, upon nucleotide binding there are importantconformational changes at the base of the coiled-coils near theATPase head, as seen in different structures of Rad50 (Lammenset al., 2011; Lim et al., 2011; M€ockel et al., 2012; G. J. Williamset al., 2011). Supporting this idea, the affinity of Rad50 to ATPand its hydrolysis are different when coiled-coils are truncated(Deshpande et al., 2014). In the next section, we explore how thesechanges in the ATPase head of Rad50 occur.

5. Functional dynamics of the Mre11-Rad50 ATPase headcomplex

Dynamic, ATP-dependent conformational changes in the Rad50ATPase domain controls Mre11 nuclease activity and ATM signaling.In the absence of ATP, Mre11-Rad50 is in an open state, allowingaccess to the Mre11 active site (Fig. 4A). A crystal structure of abacterial Mre11-Rad50 homolog (SbcD-SbcC) revealed a striking,extended conformation exists in the crystal, and the relatively closefit of the theoretical SAXS scattering curve to experimental datasuggests that the bacterial complex is indeed in this rigid confor-mation in solution (Lammens et al., 2011). In contrast, SAXS datawith archaeal Mre11-Rad50 revealed that the ATP-free complex isflexible in solution, conformationally sampling open and closedconformations (Rambo and Tainer, 2011; Williams et al., 2011).

Fig. 5. Flexibility and dynamics are part of Mre11 and Nbs1's structures. Top: Structure of MRN highlighting flexible segments (arrows). Color-coding is the same as Fig. 1, exceptRad50 is gray and the capping domain of Mre11 is cyan. Bottom: schematic of Mre11 and Nbs1's sequences with arrows depicting segments of flexibility and dynamics. Dashed linesand arrows represent intrinsically disordered segments.

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Such differences may be due to differing cellular roles of bacterialversus archaeal Mre11-Rad50 complexes. Flexibility within Mre11-Rad50 likely stems from a linker that connects the Mre11 phos-phodiesterase and capping domains to a C-terminal Rad50 bindingdomain, which binds to the base of the Rad50 coiled coils allowingRad50 subunits to move with respect to the Mre11 dimer (Williamset al., 2011).

Upon nucleotide binding, Rad50 head domains dimerize andblock the Mre11 active site, as revealed by crystal structures ofbacterial Mre11-Rad50 (M€ockel et al., 2012) and archaeal Mre11-Rad50 (Lim et al., 2011) (Fig. 4A). SAXS results reveal that thisATP-bound Mre11-Rad50 complex is in a compact, rigid confor-mation (Rambo and Tainer, 2011; Williams et al., 2011), with theo-retical scattering curves fitting experimental data remarkably well(Deshpande et al., 2014). In addition to relatively small conforma-tional changes in Mre11 (Lim et al., 2011; Schiller et al., 2012), ATP-binding by Rad50 is coupled to a large conformational changewithin each Rad50monomer, with the N-lobe rotating ~35� relativeto the C-lobe. This conformational change allows the Walker A boxof one subunit and the signaturemotif plus theWalker Bmotif of theother subunit to come together to form the active sites that sand-wich two ATP molecules at the dimer interface (Hopfner et al.,2000). Furthermore, concerted changes in two coupling helicesare involved in repositioning the base of the Rad50 coiled-coils andattached Mre11 Rad50 binding domain (RBD), which may beinvolved in regulating Mre11 activities (Williams et al., 2011).

The switch between closed and open forms of Mre11-Rad50 iscontrolled by Rad50 ATP hydrolysis and product release, which inturn controls the nuclease activity of Mre11 (Majka et al., 2012).Cross-linking experiments verified that in the ATP-bound closedstate, Mre11 nuclease activity is switched off. Furthermore, openingof the Rad50 dimer following ATP hydrolysis is required for bothMre11 endonuclease and exonuclease activities (Deshpande et al.,2014). Interestingly, Rad50 has a slow ATP hydrolysis rate (Majkaet al., 2012). Moreover, elegant biochemical analysis of a T4Rad50 homolog revealed that the chemistry of ATP hydrolysis islikely the rate-limiting step (Herdendorf et al., 2011; Herdendorfand Nelson, 2014), meaning that within the cell this ATP-boundform is likely to exist for a substantial amount of time before ATPhydrolysis and subsequent release of ADP and phosphate returnsthe complex to the open state for another round of the ATPase cycle.Further insights into the biological importance of open and closedstates of Mre11-Rad50 and the rate of Rad50 ATPase activityemerged from investigations into point mutations designed todisrupt ATP-dependent Rad50 conformations (Deshpande et al.,2014), as discussed in more detail below.

The molecular environment surrounding the extended signa-ture motif, which includes the a-helix following the classical ABC-ATPase signature motif, facilitates the plastic deformations neces-sary for ATP-dependent conformational changes (Fig. 4B). Theextended signaturemotif is at the intersection of the N- and C-lobesof Rad50 that undergo the 35� rotation, and contains arginine

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residues that switch between different salt-bridge networks tocoordinate subdomain rotation (Williams et al., 2011). Further-more, adjacent to the extended signature helix is an internal pro-tein cavity that is remodeled between ATP-free and ATP-boundstates, facilitating the conformational change (Deshpande et al.,2014). To further dissect the role of different Rad50 ATP-states,mutations that either disrupted a key salt-bridge networkinvolved in coordinating conformational changes (Arg805Glu inPyrococcus furiosus Rad50, and equivalent conserved residues ineukaryotic Rad50) or partially filled the protein cavity (Leu802Trpin P. furiosus Rad50, and equivalent conserved residues in eukary-otic Rad50) were studied (Deshpande et al., 2014). Biochemical andbiophysical results, including novel time-course SAXS experiments,revealed that these mutations result in distinct separation-of-functions that alter the rate at which Rad50 ATPase activityswitches the Mre11-Rad50 complex from ATP-bound and closed toATP-free and open conformations. The Arg805Glu mutationenhanced the closed ATP-bound conformation; resulting inreduced ATPase and nuclease activities, yet increased the bindingand tethering of DNA ends. In vivo assays showed that this variantwas more proficient at NHEJ than wildtype Rad50. In contrast,while the Leu802Trp mutation reduced ATP-binding, when ATPbound it was hydrolyzed more quickly than wildtype; resulting inmore time spent in the open conformation and enhanced Mre11nuclease and end-resection activities. Survival assays followingclastogen exposure, as well as DSB signaling assays, revealed thatboth mutations were deficient in DSBR and ATM activation,showing the time Rad50 spends in the ATP-bound state is criticalfor MRN functions. Interestingly, this ATP-bound state has beenimplicated in unwinding DNA ends (Cannon et al., 2013), and isrequired for MRN-mediated ATM activation in vitro (Lee et al.,2013). Recently, a crystal structure of bacterial Rad50 in complexwith DNA and ATP has been solved (Rojowska et al., 2014). Thisrevealed a biologically important Rad50-DNA binding interface thatis necessary for telomere maintenance (through ATM activation),but has little impact on repair activities (Rojowska et al., 2014).However, molecular mechanisms for how ATP-boundMre11-Rad50unwinds DNA ends, activates ATM, and hands off DNA from Rad50to Mre11 for nucleolytic processing remain enigmatic.

The results using Rad50 mutations imply that modulatingRad50's ATPase activity can affect biological outcomes by favoringHRR versus NHEJ or by impacting DSB signaling. Furthermore, thelong-range allostery observed for MRN suggests that alterations tothe Zn-hook and/or the coiled-coils of Rad50 or of Mre11/Nbs1subunits can impact the ABC-ATPase domain. This collective dataraises the intriguing possibility that in eukaryotes post-translational modification within the MRN complex or proteinpartner interactions may alter Rad50 ATPase activities to controlrepair pathway choice and/or signaling depending on the biologicalcontext. With the progress in SAXS experiments and analysis thatcan provide accurate structures (Putnam et al., 2007; Schneidman-Duhovny et al., 2013), comprehensive conformations (Hura et al.,2013a; Rambo and Tainer, 2010), high throughput determinationof the precise mass of assemblies in solution (Hura et al., 2009;Rambo and Tainer, 2013a), and the ability to track DNA conforma-tions with gold nanocrystals (Hura et al., 2013b), we can expectfurther results on the functional flexibility and conformations ofRad50 and MRN.

6. Mre11 conformations act in Rad50's ATP-dependentdimerization and DNA sculpting

We now have a good sampling of Mre11 catalytic domainstructures from several different organisms in the Archaea(Hopfner et al., 2001; Lim et al., 2011), Bacteria (Das et al., 2010; Liu

et al., 2014) and Eukarya kingdoms (Park et al., 2011; Schiller et al.,2012). They all have a common fold, with the phosphodiesterasecore and capping domains, and their active site arrangement is alsoconserved. However, the dimer organization is different in allstructures, which highlights the flexible nature of Mre11. Thedimerization of Mre11 is crucial for stable DNA binding (Williamset al., 2008) and differential orientations of both dimers could bea way to control and limit the nuclease activity. Moreover, recentMre11-DNA complex structures served as a basis to hypothesizethat dimer rotation could help DNA melting by twisting the duplex(Sung et al., 2014).

The role of Mre11 in melting dsDNA is also hypothesized in amodel of its enzymatic mechanism (Williams et al., 2008). In athree-step exonuclease substrate recognition mechanism, Mre11sculpts the DNA using several flexible DNA binding motifs of theprotein. More precisely, the first step is the interaction of the Mre11dimer with the phosphates and minor groove of duplex DNA (Sunget al., 2014; Williams et al., 2008). Then, the inter-subunit orien-tation changes, the capping domain rotates and a helical wedgeinserts into the DNA minor groove, which causes the DNA end tomelt. The scissile strand is then directed towards the cappingdomain, a necessary step to control and identify the substrate.Finally, a conserved histidine facilitates phosphate rotation thatdirects the scissile strand into the Mre11 active site for 30 to 50

exonucleotytic cleavage, which proceeds in a processive manner.Mutating this histidine has an effect on the exonuclease activity ofMre11, but not its endonuclease activity, showing that both activ-ities have different mechanisms. For the endonuclease mechanism,it is hypothesized that there is an ssDNA-binding pocket on theother side of the active side. Furthermore, since ssDNA is moreflexible than dsDNA, the histidine critical for exonuclease activity isdispensable as there is no need for substrate rotation. These hy-pothesized mechanisms have proven to be useful models, yet aMre11-DNA structure with DNA trapped in the active site wouldprovide further molecular insights into Mre11 activities. Currently,the ability of Mre11 to sculpt DNA for specific excision is consistentwith a theme for structure-specific DNA and RNA nucleases, such asthe replication and repair 50 flap nuclease FEN1 (Tsutakawa et al.,2011), the structurally non-homologous apurinic/apyrimidinic en-donucleases APE1 and endonuclease IV (Tsutakawa et al., 2013), aswell as multiple other structure-specific nucleases (Tsutakawaet al., 2014).

The differences in the hypothesized endonuclease versusexonuclease mechanisms were exploited for the development ofinhibitors specific to each activity of Mre11 (Shibata et al., 2014).Several inhibitors were discovered using the structure of Thermo-toga maritimaMre11 with the Mre11 inhibitor Mirin as a guide, andexploiting a focused chemical library. PFM01 and PFM03 inhibit theendonuclease activity of Mre11 by blocking the hypothesizedssDNA-binding groove, while PFM39 binds near the histidineinvolved in phosphate rotation to inhibit the exonuclease activity.Both types of inhibitors reduce DSB end resection in vivo, but createdistinct phenotypes. Inhibition of the endonuclease activity directsDSBR towards NHEJ instead of HRR, whereas exonuclease inhibitioncauses a repair defect. This repair defect could be partially rescuedby knock-out of EXO1/BLM in conjunction with exonuclease inhi-bition, which phenocopies endonuclease inhibition by pushingrepair towards NHEJ. Collectively, these results showed that Mre11endonuclease acts upstream of exonuclease and supports a two-step model of Mre11 activity in end resection for HRR. First,Mre11 uses its endonuclease activity to nick duplex DNA dozens ofbase pairs away from the break. Second, Mre11 exonuclease digeststhe DNA back towards the DNA break, with BLM helicase workingtogether with either EXOI nuclease or DNA2 nuclease/helicase todigest DNA 50e30 (Nimonkar et al., 2011; Niu et al., 2010), in order to

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generate long single-strand DNA overhangs and to commit thepathway choice towards HRR (Fig. 2). These inhibitors are impor-tant tools to study pathway selection, since there is no knownmutation of Mre11 impacting only the endonuclease activity.

Besides its importance in break repair, Mre11 acts along withBRCA1, BRCA2/FANCD1 and the ATPase SMARCAL1 to protect ordegrade replication forks (Mason et al., 2014; Schlacher et al., 2011,2012), consistent with its crystal structures with two DNA ends or aY-DNA mimic for a replication fork (Williams et al., 2008). Mre11furthermore provides a backup removal of covalently attachedproteins at the replication fork (Lee et al., 2012), which is also afunction of TDP2 (Shi et al., 2012). While the mechanistic basis forMre11 function at the replication fork is poorly understood, char-acterizing distinct roles of Mre11 versus other nucleases in thiscontext will be important keys to understanding replication fidelity.

We further hypothesize that flexibility of Mre11 has a role inregulation. Indeed, the flexibility of the Mre11 dimer appears to behigher in eukaryotes, as highlighted by stabilization of Schizo-saccharomyces pombe Mre11 dimers by Nbs1 interaction (Schilleret al., 2012). Indeed, in structures of Mre11 with Nbs1, two Nbs1molecules interact with theMre11 dimer (Schiller et al., 2012). Only14 residues of Nbs1 are involved in this interaction (Fig. 5). Inter-estingly, a 7-residue segment from one Nbs1 molecule is makingcontacts with both Mre11 subunits simultaneously, stabilizing theMre11 dimer. The interaction site is on the opposite surface ofMre11 to its active site and has flexible loops unique to eukaryotes.The binding of Nbs1 on this site induces an ordering of the loopsand a 30� rotation of the Mre11 dimer, which could have an impacton bound Rad50 or interactions with other proteins such as ATM orCtIP. Such flexibility may provide a target for the development ofinhibitors by finding ligands that bind and stabilize an inactiveconformation of Mre11 dimer. In the next section we explore howflexibility of MRN proteineprotein interactions regulates pathwaychoices.

7. Nbs1 and CtIP regulate pathway choice

Nbs1 can be viewed as a sensor as well as an effector. Through itsinteraction across the Mre11 dimer interface, it senses the confor-mation of Mre11, which in turn is impacted by Rad50 ATP-state. Thebiological importance of this is still unclear, but we know thatmutations in Nbs1 can increase the nuclease activity of Mre11during replication and thus increases the number of DSBs (Crownet al., 2013). Another biological role of the Nbs1-Mre11 interac-tion could be linked with ATM signaling (Lee and Paull, 2005,2004). Indeed, the Mre11-interacting segment of Nbs1 is adjacentto the ATM-interaction motif, located at the C-terminus of theprotein (You et al., 2005). To support that model, Nbs1 and itsconnection of MRN to ATM are able to regulate the Mre11 nucleasebased upon an examination of Mre11 activity in Ataxia-telangiectasia cells (Rahal et al., 2010). Furthermore, mutations ofMre11 can dissect DSBR and ATM activation functions revealing keyroles for Nbs1 partner Mre11 in both processes (Limbo et al., 2012).Modulation of Mre11-Rad50 conformation could be sensed byNbs1, which in turn recruits ATM for its activation.

A second sensor interface of Nbs1 is its ordered N-terminaldomain,which interactswith different proteins, and thus senses thedamage status of the cell (Williams et al., 2009). More specifically itinteracts with disordered segments of proteins, and senses theirphosphorylated form. Its two phosphoprotein-binding sites (one onthe FHA domain and one on the BRCT-repeat domain) confersseveral binding modes (R. S. Williams et al., 2009). CtIP (Lloyd et al.,2009; Williams et al., 2009) MDC1 (Hari et al., 2010; Lloyd et al.,2009; Spycher et al., 2008), ATR (Olson et al., 2007), and WRN(Cheng et al., 2004; Kobayashi et al., 2010) have been shown to

interact directly with Nbs1. Notably, the structure of Nbs1 with aCTP1-based phosphopeptide (ortholog of CtIP in S. pombe) has beensolved and informs how Nbs1 acts as a sensor (R. S. Williams et al.,2009). CTP1 binding induces a long-range conformational change inthe N-terminal domain of Nbs1, which alters the interface betweenBRCT1 and BRCT2. This suggests a cross-talk between the twophosphoprotein-binding sites, which could modulate the interac-tionwith the other proteinpartners. Thismechanism could be awayfor Nbs1 to coordinate the repair pathway choice.

Beyond its roles as a sensor, Nbs1 also recruits proteins to theDNA damage site, for them to achieve their function. This includesCtIP, which has been at the center of a lot of attention and debaterecently, with its precise role and function being unclear. Unfor-tunately, no structures are currently available that could help usunderstand its mechanism. Some groups have suggested that CtIPhas an intrinsic endonuclease activity (Makharashvili et al., 2014; H.Wang et al., 2014a), which agrees with the bidirectional resectionmodel presented in Section 3. Also agreeing with that model it wasshown that Sae2 (CtIP ortholog in Saccharomyces cerevisiae) stim-ulates the endonuclease activity of MRN, but has no intrinsicendonuclease activity (Cannavo and Cejka, 2014). A possible modelis that CtIP is physically modulating the structure of MRN in orderto stimulate Mre11's endonuclease activity.

8. Clinical and therapeutic opportunities

Recently, cancer centers and the US National Cancer Institute(NCI) have initiated research programs around “failed” clinical trialsof anti-cancer drugs (Mullard, 2014). These trials were rejected,since the drug was not reducing the cancer's progression in mostpatients. But some patients responded very well to the drug andwere cured, thus they are named “exceptional responders”. Bystudying them, researchers hypothesize that they can tailor drugregimes specifically for individual patients, the idea behindpersonalized medicine. If we know why those people are excep-tional responders using genomics, whole-genome sequencing orbiomarkers, we will be able to apply the same remedy to patientsbearing the same genotype.

A great example out of this approach is the discovery of anexceptional responder in the clinical trial of a CHK1 kinase inhibitorcombined with the DNA-damaging agent irinotecan (Al-Ahmadieet al., 2014). CHK1 is a substrate of ATR in the DNA damageresponse. Briefly, whole-genome sequencing of the tumor beforechemotherapy identified the RAD50 mutant Leu1237Phe as themutation responsible for the exceptional response. Leu-1237 is partof the D-loop of RAD50, which is close to theWalker B box, involvedin ATP hydrolysis. It is also located at the dimer interface and theside chain of the conserved residue in archaeal Rad50 switchesbetween a buried and solvent-exposed position in the dimer andmonomer crystal structures, respectively. The Leu1237Phe mutantcould thus be deficient in dimerization or in its rate of ATP hy-drolysis, processes intrinsically linked. Further biochemical char-acterization is necessary to conclude the precise impact of thismutation, but Rad50-Leu1237Phe is deficient in ATM signaling (Al-Ahmadie et al., 2014).

In considering the biophysical data, we herein suggest anotherapproach that uses the knowledge derived from exceptional re-sponders. The precise molecular understanding of the deficientmutated protein could be used to design drugs that mimic themutation. Such drugs, when used in combination with other ther-apies, would work for a wide range of patients. For example, if wecould design a drug against Rad50 that mimics the effect of theLeu1237Phe mutation, combined with irinotecan and the CHK1inhibitor, this drug would be successful for most patients havingthe same cancer, not to only a subset of them.

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9. Combining biophysics and molecular biology tounderstand MRN's structural flexibility

Crystal structures of the individual MRN domains and multiplecomplexes have provided detailed knowledge for mechanistic un-derstanding. Crystallography provides precise structures but lessinformation about flexibility, although temperature factors canidentify functional flexible regions when corrected for crystalcontacts (Tainer et al., 1984). Unveiling the roles of flexibility in DNAdamage responses is an ongoing process. Flexibility for interactionand functional access to DNA ends makes sense geometrically, andis seen in systems such as the Ligase-PCNA interaction (Pascal et al.,2006). Yet, intra-molecular interactions of flexible regions cancontribute to the stability of the folded region and can act inintermolecular interaction, making interpretations of functionschallenging by single methods (Hegde et al., 2013). Thus,combining biophysical techniques together with molecularbiology will be crucial to understand flexibility of macromolecularmachines.

SAXS is an ideal biophysical technique to be combined withother structural biology techniques. Its development helped studyMre11, Rad50 (along with other members of the ABC ATPasesfamily) and Nbs1. For example, gold nanocrystal SAXS was createdto provide a means to examine DNA conformations at high sensi-tivity along a complex reaction pathway in the context of multipleprotein components (Hura et al., 2013b). Similarly, the ABC-ATPaseMutSb was used to develop a SAXS-based method to objectivelyand comprehensively compare conformational states in solution(Hura et al., 2013a). Also, we know that water molecules are part ofthe active site and other concave surface features and that residuecomposition impact bound waters (Kuhn et al., 1992, 1995). Anexciting recent observation is that our refined macromolecularcrystal structure models are in general not limited by data noise orerror but by our ability to properly model the water structure andmodel flexibility (Holton et al., 2014). We can expect that furtheradvances in biophysical methods that define flexibility and boundwater structures may allow more accurate and complete models ofdynamic complexes, including MRN.

Besides the dynamics of defined domains and complexes, it willbe important to look at the impacts of post-translational modifi-cations on MRN. Dynamic proteineprotein interactions and com-plex assemblies mediating DNA damage response are ofteninitiated or regulated by protein post-translational modification,such as phosphorylation, methylation, ubiquitination, and sumoy-lation. SAXS has proven useful to define the activity and thestructural impacts of these modifications (Datta et al., 2009; Dudaet al., 2008; Konarev et al., 2014). Indeed, SAXS and molecularbiology showed that the ubiquitin ligase RNF4 selectively recog-nizes and ubiquitinates of SUMO-modified histones, therefore itintegrates signaling by SUMO and ubiquitin (Groocock et al., 2014).We know from SAXS analyses of NHEJ that phosphorylation ofDNA-PKcs causes a functionally important conformational changeto promote opening and release from DNA (Hammel et al., 2010).We can expect to see analogous conformational regulation for MRNconformations and flexibility (Deshpande et al., 2014). Indeed, in-dependent modes of MRN phosphorylation have been reported,with CDK1 orthologs phosphorylating MRN during mitosis and,independently, ATM orthologs phosphorylating MRN after DNAdamage (Martin et al., 2014; Simoneau et al., 2014). This suggeststhat MRN integrates signals from an intricate network of phos-phoregulatory mechanisms to modulate DSB responses. Further-more, Rad17 phosphorylation by ATM at a novel Thr622 site resultsin its interaction with NBS1, facilitating recruitment of the MRNcomplex and ATM to the DSB, thereby enhancing ATM signaling (Q.Wang et al., 2014b).

Crystal structures have also revealed important results per-taining to flexibility and activity by defining the metal bindingsites in Mre11 and Rad50 domains. Frequently, metal ions are anover-looked issue in characterizing macromolecular complexes.Mre11 and Rad50 bind multiple metal ions; so far zinc, magne-sium, and manganese have been defined crystallographically.Identifying and characterizing the roles of metal ions is oftenchallenging, in part because they may not be correctly incorpo-rated into the protein in recombinant expression hosts, and maythus require purification from native biomass (Cvetkovic et al.,2010; Yannone et al., 2012). Even partial loss of metal ions im-pacts proper function and increases flexibility and misassembly,as seen for the stress response enzyme superoxide dismutase(Pratt et al., 2014) and the DNA repair and transcription helicaseXPD (Fan et al., 2008). Biophysical techniques allowing the accu-rate identification and measurement of metal ions will beimportant in future research.

One thing that can guide flexible enzyme-substrate recognitionand allows flexibility of the enzyme's binding site is the negativelycharged DNA and an appropriate positive patch on the repairenzyme surface (Huffman et al., 2005; Mol et al., 2000; Slupphauget al., 1996). Such positive patches are seen on both Mre11 andRad50 (Hopfner et al., 2001) and are consistent with subsequentDNA-complex crystal structures (Rojowska et al., 2014; Williamset al., 2008). Indeed electrostatics are part of DNA mimicry thatcan regulate DNA repair processes (Mol et al., 1995; Putnam et al.,1999; Putnam and Tainer, 2005). In general, electrostatic potentialgradients can increase the rate of interactions (Getzoff et al., 1983)as well as properly orient two macromolecules for productive col-lisions (Roberts et al., 1991). In the future it will be interesting tounderstand how the distinct Mre11 and Rad50 DNA binding sitescooperate to bind and then process DNA substrates.

10. Perspective and conclusion

In eukaryotic cells, the initiation of end resection has emergedas a critical regulatory step to differentiate between homology-dependent and end-joining repair of DSBs and to impact replica-tion fork protection versus replication fork excision. MRN is the keycomplex in these two essential components of cell biology. There-fore, a bottom up mechanistic understanding is worthwhile for itsbiological and medical impacts.

Since the first human DNA repair enzymes revealed DNAbending and nucleotide flipping in damage recognition(Slupphaug et al., 1996), structural biology has revealed confor-mational changes and flexibility as a theme in recognition speci-ficity. With MRN we have come to appreciate how the dynamicsand kinetics of the system can modulate distinct outcomes. Mre11and Rad50 both sculpt the DNA to achieve specificity as seen forbase repair enzymes (Slupphaug et al., 1996), nucleotide excisionrepair enzymes (Fuss and Tainer, 2011) and proteins that provideconnections between DNA repair pathways (Dalhus et al., 2013).Going forward we expect that hybrid methods, such as SAXScombined with crystallography or NMR, will play key roles inexamining functionally important structural flexibility in solutionunder near physiological conditions (Putnam et al., 2007; Ramboand Tainer, 2013b).

To conclude, MRN is a highly dynamic protein complex, withdifferent parts being flexible in both their structure and mecha-nism. Understanding the dynamics of the MRN complex is helpingdrive novel and combined biophysical methods and is furthermoreessential for the detailed knowledge necessary to both understandand predict biological responses to DSBs, and for the developmentof novel medical approaches to cancer.

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Acknowledgments

Work for this review and the authors' efforts on the MRNcomplex were funded by NIH R01CA117638 and P01CA092584.J.L.V. is recipient of a fellowship from the Canadian Research In-stitutes of Health Research (CIHR).

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