Rescuing stalled replication forks

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Mar 10, 2011 - tion forks, suggesting that MMS22L is particularly important to repair DSBs. Rescuing stalled replication forks. MMS22L-TONSL, a novel ...

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Cell Cycle 10:11, 1703-1705; June 1, 2011; © 2011 Landes Bioscience

Rescuing stalled replication forks

MMS22L-TONSL, a novel complex for DNA replication fork repair in human cells Wojciech Piwko,* Raymond Buser and Matthias Peter* Department of Biology; Institute of Biochemistry; Eidgenössische Technische Hochschule Zurich; Zurich, Switzerland

The eukaryotic genome is particularly susceptible to DNA damage during S phase of the cell cycle, as DNA lesions can lead to stalling and collapsing of DNA replication forks. In order to maintain genome integrity, the S-phase checkpoint protects replication forks and cooperates with specialized DNA repair pathways to prevent DNA damage and to resume DNA replication. In budding yeast, the CUL4-type cullin Rtt101 forms a multisubunit ubiquitin ligase with the adaptor proteins Mms1 and Mms22, which together support replication through natural pause sites and damaged DNA templates.1 Yeast cells lacking either of these proteins are specifically deficient in homologous recombination (HR)-mediated restart of replication from damaged DNA templates.2,3 The organization of the Rtt101 complex implies that Mms22 may serve as a substrate specificity adaptor that recognizes and ubiquitylates proteins at stalled forks in order to restart replication. Recently, four independent research groups using distinct functional assays and homology searches identified a putative human homolog of Mms22, MMS22L.4-7 RNAi-mediated depletion of MMS22L leads to arrest of cells in G2-phase as a consequence of accumulating spontaneous DNA damage during S phase. In contrast to their resistance to ionizing radiation, MMS22L-depleted cells are strongly sensitive to camptothecin, which induces double-strand breaks (DSBs) by interfering with progression of replication forks, suggesting that MMS22L is particularly important to repair DSBs

resulting from collapsed replication forks. Several experimental approaches revealed that MMS22L is involved in regulation of HR-mediated repair, a process implicated in restarting stalled/collapsed replication forks. Specifically, MMS22L promotes displacement of single-stranded DNA (ssDNA)-binding protein RPA and assembly of RAD51 filaments at camptothecin-induced damage sites,4,6 implying that it regulates the strand invasion step of HR (Fig. 1). Thus, MMS22L appears critical for dealing with replication fork stalling/collapsing events and protects cells from DSB formation during S phase. In support of this hypothesis, DNA combing experiments confirmed that replication fork speed is reduced in MMS22L-depleted cells,7 and revealed specific defects in restart of replication forks after camptothecin-mediated replication stress.6 Overall, loss of MMS22L leads to strikingly similar phenotypes as those observed in yeast strains lacking Mms22, suggesting that both proteins act in an evolutionarily conserved pathway regulating replication fork progression during normal and perturbed S phase to ensure genome stability. Proteomic analysis of MMS22Lassociated proteins revealed a direct interaction with NFKBIL2/TONSL, a poorly characterized protein related to Tonsoku/ BRU1, which is involved in genome stability maintenance in A. thaliana.8 Indeed, depletion of TONSL phenocopies depletion of MMS22L, indicating they form a novel DNA damage repair complex. Moreover, both proteins accumulate at sites of replication stress/DNA damage  

induced by camptothecin or UV laser and knocking-down MMS22L abolishes recruitment of GFP-TONSL,6 suggesting that MMS22L is required for targeting the complex to collapsed replication forks. Owing to its multi-protein interaction domain structure, TONSL functions as a scaffolding component responsible for assembling a larger DNA repair complex. Indeed, it binds to several components of DNA replication and repair machineries, including MCM replicative helicase subunits and the histone chaperones FACT, ASF1A and ASF1B, implicating the MMS22L-TONSL complex in regulating histone deposition at replication forks. However, how nucleosome assembly pathways impact MMS22L-TONSL-mediated repair processes awaits further analysis. Importantly, the MMS22L-TONSL homologs—yeast Mms22 and plant BRU1—are linked to chromatin modification and remodeling pathways, and these components seem to be required for their functions in genome stability maintenance.8,9 Altogether, these findings suggest that the MMS22L-TONSL complex acts directly at replication forks, where MMS22L facilitates the recruitment of the complex to recombination substrates at collapsed replication forks, while TONSL promotes a HR-permissive chromatin organization, stimulating assembly of RAD51 filaments (Fig. 1). The functional similarity between human MMS22L and yeast Mms22 raises the question of their molecular homology. Both proteins share only weak local homology and a cursory review suggests they promote genome stability by

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*Correspondence to: Matthias Peter and Wojciech Piwko; Email: [email protected] and [email protected] Submitted: 03/10/11; Accepted: 03/20/11 DOI: 10.4161/cc.10.11.15557 Comment on: Piwko W, et al. EMBO J 2010; 29:4210–22; Duro E, et al. Mol Cell 2010; 40:632–44; O’Connell BC, et al. Mol Cell 2010; 40:645–57; and O’Donnell L, et al. Mol Cell 2010; 40:619–31. www.landesbioscience.com

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Figure 1. Model of the function and regulation of the MMS22L-TONSL repair complex at stalled DNA replication forks. (A) The complex binds histone chaperones (FACT, ASF1A and ASF1B) and may promote replication by regulating histone deposition. (B) TONSL localizes to sites of ongoing DNA replication by interaction with the replicative helicase, while MMS22L is recruited to regions of ssDNA generated by replication fork stalling/collapse or DSB end resection. We speculate that the MMS22L-TONSL complex promotes HR-dependent repair/restart of replication at these sites by stimulating displacement of RPA and/or loading of RAD51, and likely by orchestrating chromatin remodeling.

forming distinct molecular complexes. Unlike the yeast counterpart, human MMS22L does not assemble into a stable ubiquitin ligase complex. However, MMS22L is degraded by the proteasome upon replication stress and by a CUL4DDB1-dependent mechanism.7 Although the functional importance of this degradation is not clear, a similar mechanism is implicated in completion of DNA repair in yeast cells.10 While TONSL has no obvious yeast homologues, Esc2 interacts with Rtt101,11 and possesses a

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unique ubiquitin-like domain similar to the one found in TONSL. Indeed, Esc2 is involved in regulating replication-associated recombinational repair possibly by chromatin structure modification,12 raising the possibility that it may function as the yeast counterpart of TONSL. The identification of the MMS22LTONSL complex represents an important step towards understanding how eukaryotic cells channel the repair processes to resolve specific DNA damage problems. It will now be important to identify the

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specific lesions/HR-promoting structures recognized by MMS22L and to understand the molecular activities associated with the MMS22L-TONSL complex. Interestingly, GFP-TONSL is recruited to UV irradiation-induced stripes independently of the cell cycle phase,4 raising the possibility that MMS22L/TONSLregulated activities may not be restricted to DNA replication forks. It is also possible that TONSL acts independently of MMS22L to repair certain types of damage, e.g., by associating with a DNA

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damage recognition component distinct from MMS22L. Indeed, our proteomics approach revealed that KU70-KU80 associates with TONSL and could thus promote non-homologous end joining. Undoubtedly, additional studies are required to reveal the dynamics of MMS22L-TONSL complex assembly during the cell cycle and in response to various types of DNA damage.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Luke B, et al. Curr Biol 2006; 16:786-92. Zaidi IW, et al. EMBO Rep 2008; 9:1034-40. Duro E, et al. DNA Repair (Amst) 2008; 7:811-8. Duro E, et al. Mol Cell 2010; 40:632-44. O’Connell BC, et al. Mol Cell 2010; 40:645-57. O’Donnell L, et al. Mol Cell 2010; 40:619-31. Piwko W, et al. EMBO J 2010; 29:4210-22. Takeda S, et al. Genes Dev 2004; 18:782-93. Collins SR, et al. Nature 2007; 446:806-10. Ben-Aroya S, et al. PLoS Genet 2010; 6:1000852. Mimura S, et al. J Biol Chem 2010; 285:9858-67. Mankouri HW, et al. Mol Biol Cell 2009; 20:1683-94.

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