David T. Long

Mechanism of RAD51-Dependent DNA Interstrand Cross-Link Repair

An in vitro system reveals the steps involved in repairing covalent links between DNA strands of the double helix. DNA interstrand cross-links (ICLs) are toxic DNA lesions whose repair in S phase of eukaryotic cells is incompletely understood. In Xenopus egg extracts, ICL repair is initiated when two replication forks converge on the lesion. Dual incisions then create a DNA double-strand break (DSB) in one sister chromatid, whereas lesion bypass restores the other sister. We report that the broken sister chromatid is repaired via RAD51-dependent strand invasion into the regenerated sister. Recombination acts downstream of FANCI-FANCD2, yet RAD51 binds ICL-stalled replication forks independently of FANCI-FANCD2 and before DSB formation. Our results elucidate the functional link between the Fanconi anemia pathway and the recombination machinery during ICL repair. In addition, they demonstrate the complete repair of a DSB via homologous recombination in vitro.

Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

Uncrossing covalently linked DNA strands DNA interstrand cross-links (ICLs) covalently link the two strands of the double helix. ICL mutations are difficult to repair, because the two DNA strands cannot be separated and so one strand cannot be used as a template to repair the other. Räschle et al. developed a mass spectrometry–based method to systematically analyze a time series of all the proteins recruited to repair ICLs in Xenopus egg extracts. They found many of the known factors required for ICL repair. They also found a number of new factors, two of which define a new repair pathway for ICL mutations. Science, this issue 10.1126/science.1253671 Surveying the battery of proteins required to repair covalently linked DNA strands reveals a new repair pathway. INTRODUCTION DNA damage encountered during DNA replication represents a major challenge to the integrity of the genome. Because replicative polymerases are unable to synthesize across DNA lesions, prolonged stalling of replisomes can lead to replication fork collapse, giving rise to gross genomic alterations. Cells have evolved intricate responses that orchestrate the reorganization of the replication fork necessary for overcoming such roadblocks, but the full set of factors involved in these processes has not been defined. Here, we performed unbiased proteomic analyses of the dynamically changing protein landscape at damaged chromatin undergoing DNA replication. This yielded mechanistic insights into the pathways that ensure genomic stability during perturbed DNA replication. RATIONALE We combined the powerful and well-established Xenopus egg extract system for cell-free DNA replication with quantitative mass spectrometry to develop CHROMASS (chromatin mass spectrometry), a simple yet robust method for the unbiased analysis of chromatin composition. Using bifunctional cross-linkers, compounds commonly applied in chemotherapy, we systematically monitored the assembly and disassembly of protein complexes on replicating chromatin containing DNA interstrand cross-links (ICLs). RESULTS We show that replication of ICL-containing chromatin templates triggers recruitment of more than 90 DNA repair and genome maintenance factors. Addition of replication inhibitors revealed the subset of proteins that accumulate in a strictly replication-dependent fashion. The quantitative readout by CHROMASS is highly lesion-specific, as the known repair factors enriched on psoralen–cross-linked templates had previously been linked to ICL repair or specific branches of DNA damage signaling. In contrast, virtually none of the proteins involved in unrelated DNA repair pathways (e.g., base excision repair or nonhomologous end joining) showed damage-specific enrichment. The temporal profiles of hundreds of proteins across an extensive time course and a variety of perturbations provided a data-rich resource that could be mined to identify previously unknown genome maintenance factors. Among such hits, we identified SLF1 and SLF2 and showed that they physically link RAD18 with the SMC5/6 complex. This defines a linear RAD18-SLF1-SLF2 recruitment pathway for the SMC5/6 complex to RNF8/RNF168-generated ubiquitylations at damaged DNA in vertebrate cells. We found that SLF2 is a distant ortholog of yeast NSE6, an SMC5/6-associated factor that is essential for targeting this complex to damaged DNA to promote faithful repair of the lesions. Consistent with pivotal functions of SMC5/6 in the suppression of replication stress-induced, illegitimate recombination intermediates, depletion of SLF1 or SLF2 led to mitotic errors and compromised cell survival in response to genotoxic agents. CONCLUSIONS CHROMASS enables rapid and unbiased time-resolved insights into the chromatin interaction dynamics of entire DNA repair pathways. Combined with specific perturbations, CHROMASS allows systems-level interrogation of the consequences of inactivating particular aspects of the repair process. We compiled comprehensive proteome-wide profiles of dynamic protein interactions with damaged chromatin. These can be mined to pinpoint genome stability maintenance factors, exemplified here by the identification of SLF1 and SLF2, which define a recruitment pathway for the SMC5/6 complex. CHROMASS can be applied to other chromatin-associated pathways and may also shed light on the dynamics of posttranslational modifications governing the regulation of these processes. CHROMASS analysis of proteins recruited to stalled replication forks reveals a specific set of DNA repair factors involved in the replication stress response. Among these, SLF1 and SLF2 are found to bridge the SMC5/6 complex to RAD18, thereby linking SMC5/6 recruitment to ubiquitylation products formed at various DNA lesions. DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells.

BRCA1 Promotes Unloading of the CMG Helicase from a Stalled DNA Replication Fork

The tumor suppressor protein BRCA1 promotes homologous recombination (HR), a high-fidelity mechanism to repair DNA double-strand breaks (DSBs) that arise during normal replication and in response to DNA-damaging agents. Recent genetic experiments indicate that BRCA1 also performs an HR-independent function during the repair of DNA interstrand crosslinks (ICLs). Here we show that BRCA1 is required to unload the CMG helicase complex from chromatin after replication forks collide with an ICL. Eviction of the stalled helicase allows leading strands to be extended toward the ICL, followed by endonucleolytic processing of the crosslink, lesion bypass, and DSB repair. Our results identify BRCA1-dependent helicase unloading as a critical, early event in ICL repair.

The MCM8-MCM9 Complex Promotes RAD51 Recruitment at DNA Damage Sites To Facilitate Homologous Recombination

ABSTRACT The minichromosome maintenance protein homologs MCM8 and MCM9 have previously been implicated in DNA replication elongation and prereplication complex (pre-RC) formation, respectively. We found that MCM8 and MCM9 physically associate with each other and that MCM8 is required for the stability of MCM9 protein in mammalian cells. Depletion of MCM8 or MCM9 in human cancer cells or the loss of function MCM9 mutation in mouse embryo fibroblasts sensitizes cells to the DNA interstrand cross-linking (ICL) agent cisplatin. Consistent with a role in the repair of ICLs by homologous recombination (HR), knockdown of MCM8 or MCM9 significantly reduces HR repair efficiency. Chromatin immunoprecipitation analysis using human DR-GFP cells or Xenopus egg extract demonstrated that MCM8 and MCM9 proteins are rapidly recruited to DNA damage sites and promote RAD51 recruitment. Thus, these two metazoan-specific MCM homologs are new components of HR and may represent novel targets for treating cancer in combination with DNA cross-linking agents.

Fork regression is an active helicase‐driven pathway in bacteriophage T4

Reactivation of stalled replication forks requires specialized mechanisms that can recognize the fork structure and promote downstream processing events. Fork regression has been implicated in several models of fork reactivation as a crucial processing step that supports repair. However, it has also been suggested that regressed forks represent pathological structures rather than physiological intermediates of repair. To investigate the biological role of fork regression in bacteriophage T4, we tested several mechanistic models of regression: strand exchange‐mediated extrusion, topology‐driven fork reversal and helicase‐mediated extrusion. Here, we report that UvsW, a T4 branch‐specific helicase, is necessary for the accumulation of regressed forks in vivo, and that UvsW‐catalysed regression is the dominant mechanism of origin‐fork processing that contributes to double‐strand end formation. We also show that UvsW resolves purified fork intermediates in vitro by fork regression. Regression is therefore part of an active, UvsW‐driven pathway of fork processing in bacteriophage T4.

The Phage T4 Protein UvsW Drives Holliday Junction Branch Migration

The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.

Regression supports two mechanisms of fork processing in phage T4

Replication forks routinely encounter damaged DNA and tightly bound proteins, leading to fork stalling and inactivation. To complete DNA synthesis, it is necessary to remove fork-blocking lesions and reactivate stalled fork structures, which can occur by multiple mechanisms. To study the mechanisms of stalled fork reactivation, we used a model fork intermediate, the origin fork, which is formed during replication from the bacteriophage T4 origin, ori(34). The origin fork accumulates within the T4 chromosome in a site-specific manner without the need for replication inhibitors or DNA damage. We report here that the origin fork is processed in vivo to generate a regressed fork structure. Furthermore, origin fork regression supports two mechanisms of fork resolution that can potentially lead to fork reactivation. Fork regression generates both a site-specific double-stranded end (DSE) and a Holliday junction. Each of these DNA elements serves as a target for processing by the T4 ATPase/exonuclease complex [gene product (gp) 46/47] and Holliday junction-cleaving enzyme (EndoVII), respectively. In the absence of both gp46 and EndoVII, regressed origin forks are stabilized and persist throughout infection. In the presence of EndoVII, but not gp46, there is significantly less regressed origin fork accumulation apparently due to cleavage of the regressed fork Holliday junction. In the presence of gp46, but not EndoVII, regressed origin fork DSEs are processed by degradation of the DSE and a pathway that includes recombination proteins. Although both mechanisms can occur independently, they may normally function together as a single fork reactivation pathway.

The ε Subunit of DNA Polymerase III Is Involved in the Nalidixic Acid-Induced SOS Response in Escherichia coli

Quinolone antibacterial drugs such as nalidixic acid target DNA gyrase in Escherichia coli. These inhibitors bind to and stabilize a normally transient covalent protein-DNA intermediate in the gyrase reaction cycle, referred to as the cleavage complex. Stabilization of the cleavage complex is necessary but not sufficient for cell killing--cytotoxicity apparently results from the conversion of cleavage complexes into overt DNA breaks by an as-yet-unknown mechanism(s). Quinolone treatment induces the bacterial SOS response in a RecBC-dependent manner, arguing that cleavage complexes are somehow converted into double-stranded breaks. However, the only proteins known to be required for SOS induction by nalidixic acid are RecA and RecBC. In hopes of identifying additional proteins involved in the cytotoxic response to nalidixic acid, we screened for E. coli mutants specifically deficient in SOS induction upon nalidixic acid treatment by using a dinD::lacZ reporter construct. From a collection of SOS partially constitutive mutants with disruptions of 47 different genes, we found that dnaQ insertion mutants are specifically deficient in the SOS response to nalidixic acid. dnaQ encodes DNA polymerase III epsilon subunit, the proofreading subunit of the replicative polymerase. The deficient response to nalidixic acid was rescued by the presence of the wild-type dnaQ gene, confirming involvement of the epsilon subunit. To further characterize the SOS deficiency of dnaQ mutants, we analyzed the expression of several additional SOS genes in response to nalidixic acid using real-time PCR. A subset of SOS genes lost their response to nalidixic acid in the dnaQ mutant strain, while two tested SOS genes (recA and recN) continued to exhibit induction. These results argue that the replication complex plays a role in modulating the SOS response to nalidixic acid and that the response is more complex than a simple on/off switch.

Construction of Plasmids Containing Site-Specific DNA Interstrand Cross-Links for Biochemical and Cell Biological Studies

Plasmids containing a site-specific DNA interstrand cross-link (ICL) are invaluable tools for the investigation of ICL repair pathways at the biochemical and cellular level. We describe a procedure for preparation of plasmid DNA substrates containing a single ICL at a specific site. The procedure is versatile, leads to reliable yields of pure DNA substrate, and is suitable for the incorporation of virtually any type of DNA lesion into plasmids.

A Novel Function for BRCA1 In Crosslink Repair

In this issue of Molecular Cell, Bunting et al. (2012) provide new evidence that BRCA1 plays an important role in DNA interstrand crosslink repair that is distinct from its established function in promoting DNA end resection during homologous recombination.