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

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.