Playing the End Game: DNA Double-Strand Break Repair Pathway Choice J. Ross Chapman, Martin R.G. Taylor, Simon J. Boulton  Molecular Cell  Volume 47, Issue 4, Pages 497-510 (August 2012) DOI: 10.1016/j.molcel.2012.07.029 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Mechanisms of DSB Formation DSBs can be generated by exogenous or endogenous causes. DSBs can be structurally distinguished as one ended (green boxes) or two ended (yellow boxes). The latter can arise directly following IR exposure, or indirectly from enzymatic processing of converging replication forks following stalling at a common lesion, such as an interstrand crosslink (ICL). One-ended DSBs are specific to S phase, forming when replication forks encounter template strand nicks and collapse, or stall and are metabolized independently of another converging fork. The mechanisms of stalled replication fork processing to generate a DSB per se are poorly understood and involve processes including endonucleolytic incision and translesion synthesis. DSBs are then repaired by NHEJ or HR: the “choice” between these processes is regulated in various different ways. The blue oval represents Spo11 and the green circle Rag1/2 (see text for more information). Molecular Cell 2012 47, 497-510DOI: (10.1016/j.molcel.2012.07.029) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Chromatin-Mediated Regulation of DSB Repair Pathway Choice (Top panel) In G1/G0 cells, oligomeric 53BP1 binds nucleosomes in DSB-flanking chromatin to mediate a chromatin state restrictive to DSB processing, favoring accurate repair by NHEJ. Restricting nuclease access may not be exclusive from transcriptional inhibition, and 53BP1 oligomerization could also promote DSB synapsis. 53BP1 enrichment may be regulated by changes in the presentation of the constitutive H4K20me2 epitope at DSB sites, and/or de novo methylation events mediated by lysine methyl-transferases (KMTs) such as PRSET8 or MMSET (reviewed in Lukas et al., 2011). (Lower panels) Following progression into S phase, BRCA1 enriched on chromatin (by similar upstream ubiquitylation events to 53BP1) promotes chromatin changes at the DSB that permit processing and HR. BRCA1/BARD1 interactors and ubiquitin editing events are likely to contribute to such alterations and may block 53BP1 nucleosome contacts. Simultaneous BRCA1-mediated protein interactions might link different factors within distinct compartments surrounding DSBs: facilitating chromatin alterations, DNA processing events, and the subsequent HR reaction, while Rap80 complex chromatin interactions might provide negative feedback. Question marks denote potential unknown proteins and molecular interactions of unresolved function. Molecular Cell 2012 47, 497-510DOI: (10.1016/j.molcel.2012.07.029) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Initiation and Regulation of DSB Resection (A) Model of MRN- and CtIP-dependent resection initiation. Mre11 endonuclease (green triangle) nicks the 5′ strand at a distance from DSBs, initiating DNA resection by Exo1 (5′-3′ directed) and Mre11 (3′-5′ directed), and counteracting Ku-dependent end protection and NHEJ. Exactly how CtIP (blue circle) regulates this processing remains unknown. (B) CDK-dependent phosphorylation regulates CtIP activity. Mre11 bridges CtIP-CDK interactions, promoting regulatory CtIP phospho-dependent interactions with known and potentially unknown proteins. Such interactions also protect CtIP from proteolysis. (C) CtIP-dependent resection is tightly controlled. CtIP chromatin interactions are dependent on a DNA-recognition (DR) motif, yet its DSB enrichment is MRN and ATM dependent. Subsequently, SIRT6-dependent CtIP deacetylation initiates CtIP-dependent resection events. Constitutive CtIP acetylation by an unknown lysine acetyl transferase (KAT) restricts this activity to a damage context. Molecular Cell 2012 47, 497-510DOI: (10.1016/j.molcel.2012.07.029) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 HR Pathway Choice HR can initiate from one- or two-ended DSBs upon RAD51 (yellow ovals) loading onto ssDNA and strand invasion. HR initiation is limited by antirecombinases, including Srs2/PARI, RECQL5, BLM, and perhaps FBH1. RAD51 clearance from dsDNA after strand invasion then allows initiation of repair DNA synthesis. One-ended DSBs may be repaired by Pol32-dependent break-induced replication (BIR). At two-ended DSBs, the extending D loop may be displaced by RTEL1 to promote SDSA or capture the second DSB end. Nucleolytic processing (black triangles) and ligation (curly black arrows) of the D loop by MUS81-EME1 as indicated can resolve the joint molecules as a crossover. Direct ligation of the extending strands to the exposed 5′ ends of the DSB (blue circles) leads to dHJ formation. dHJ dissolution by BLM-TOPOIIIα forms noncrossover products, while endonucleolytic cleavage (black triangles) by HJ resolvases such as SLX4-SLX1 and GEN1 can result in crossover or noncrossover outcomes depending on cleavage orientation (two of the four possible products are shown). Black dashed lines indicate gene conversion tracts. Molecular Cell 2012 47, 497-510DOI: (10.1016/j.molcel.2012.07.029) Copyright © 2012 Elsevier Inc. Terms and Conditions