The multifaceted roles of the Ctf4 replisome hub in the maintenance of genome integrity.

At the core of cellular life lies a carefully orchestrated interplay of DNA replication, recombination, chromatin assembly, sister-chromatid cohesion and transcription. These fundamental processes, while seemingly discrete, are inextricably linked during genome replication. A set of replisome factors integrate various DNA transactions and contribute to the transient formation of sister chromatid junctions involving either the cohesin complex or DNA four-way junctions. The latter structures serve DNA damage bypass and may have additional roles in replication fork stabilization or in marking regions of replication fork blockage. Here, we will discuss these concepts based on the ability of one replisome component, Ctf4, to act as a hub and functionally link these processes during DNA replication to ensure genome maintenance.

SnapShot: DNA repair pathways.

The genetic information stored in DNA is under continuous threat by endogenous and environmental sources of DNA damage. Cells have evolved multiple DNA repair pathways that function in overlapping manners, with principles shared across species. Here, we depict the main DNA repair pathways cells rely on, with the primary lesions they are tackling, along with key players and main DNA transactions. To view this SnapShot, open or download the PDF.

SnapShot: Tolerating replication stress.

Completion of DNA replication relies on the ability of replication forks to traverse various types of DNA damage, actively transcribed regions, and structured DNA. The mechanisms enabling these processes are here referred to as DNA damage tolerance pathways. Here, we depict the stalled DNA replication fork structures with main DNA transactions and key factors contributing to the bypass of such blocks, replication restart, and completion. To view this SnapShot, open or download the PDF.

Hot on RAD51C: structure and functions of RAD51C-XRCC3.

A new study by Longo, Roy et al. has solved the structure of the RAD51C-XRCC3 (CX3) heterodimer with a bound ATP analog, identifying two main structural interfaces and defining separable replication fork stability roles. One function relates to the ability of RAD51C to bind and assemble CX3 on nascent DNA, with an impact on the ability of forks to restart upon replication stress. The other relates to effective CX3 heterodimer formation, required for 5' RAD51 filament capping, with effects on RAD51 filament disassembly, fork protection and influencing the persistence of reversed forks.

Computed structures of core eukaryotic protein complexes.

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.

DNA helicases in homologous recombination repair.

Helicases are in the spotlight of DNA metabolism and are critical for DNA repair in all domains of life. At their biochemical core, they bind and hydrolyze ATP, converting this energy to translocate unidirectionally, with different strand polarities and substrate binding specificities, along one strand of a nucleic acid. In doing so, DNA and RNA helicases separate duplex strands or remove nucleoprotein complexes, affecting DNA repair and the architecture of replication forks. In this review, we focus on recent advances on the roles and regulations of DNA helicases in homologous recombination repair, a critical pathway for mending damaged chromosomes and for ensuring genome integrity.

Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites.

Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.

Mus81-Mms4 endonuclease is an Esc2-STUbL-Cullin8 mitotic substrate impacting on genome integrity.

The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phosphorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly)SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a replication protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.

The Mgs1/WRNIP1 ATPase is required to prevent a recombination salvage pathway at damaged replication forks.

DNA damage tolerance (DDT) is crucial for genome integrity maintenance. DDT is mainly carried out by template switch recombination, an error-free mode of overcoming DNA lesions, or translesion DNA synthesis, which is error-prone. Here, we investigated the role of Mgs1/WRNIP1 in modulating DDT. Using budding yeast, we found that elimination of Mgs1 in cells lacking Rad5, an essential protein for DDT, activates an alternative mode of DNA damage bypass, driven by recombination, which allows chromosome replication and cell viability under stress conditions that block DNA replication forks. This salvage pathway is RAD52 and RAD59 dependent, requires the DNA polymerase δ and PCNA modification at K164, and is enabled by Esc2 and the PCNA unloader Elg1, being inhibited when Mgs1 is present. We propose that Mgs1 is necessary to prevent a potentially toxic recombination salvage pathway at sites of perturbed replication, which, in turn, favors Rad5-dependent template switching, thus helping to preserve genome stability.

Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates.

Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.

The Swr1 chromatin-remodeling complex prevents genome instability induced by replication fork progression defects.

Genome instability is associated with tumorigenesis. Here, we identify a role for the histone Htz1, which is deposited by the Swr1 chromatin-remodeling complex (SWR-C), in preventing genome instability in the absence of the replication fork/replication checkpoint proteins Mrc1, Csm3, or Tof1. When combined with deletion of SWR1 or HTZ1, deletion of MRC1, CSM3, or TOF1 or a replication-defective mrc1 mutation causes synergistic increases in gross chromosomal rearrangement (GCR) rates, accumulation of a broad spectrum of GCRs, and hypersensitivity to replication stress. The double mutants have severe replication defects and accumulate aberrant replication intermediates. None of the individual mutations cause large increases in GCR rates; however, defects in MRC1, CSM3 or TOF1 cause activation of the DNA damage checkpoint and replication defects. We propose a model in which Htz1 deposition and retention in chromatin prevents transiently stalled replication forks that occur in mrc1, tof1, or csm3 mutants from being converted to DNA double-strand breaks that trigger genome instability.

Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin.

DNA damage tolerance (DDT) mechanisms facilitate replication resumption and completion when DNA replication is blocked by bulky DNA lesions. In budding yeast, template switching (TS) via the Rad18/Rad5 pathway is a favored DDT pathway that involves usage of the sister chromatid as a template to bypass DNA lesions in an error-free recombination-like process. Here, we establish that the Snf2 family translocase Irc5 is a novel factor that promotes TS and averts single-stranded DNA persistence during replication. We demonstrate that, during replication stress, Irc5 enables replication progression by assisting enrichment of cohesin complexes, recruited in an Scc2/Scc4-dependent fashion, near blocked replication forks. This allows efficient formation of sister chromatid junctions that are crucial for error-free DNA lesion bypass. Our results support the notion of a key role of cohesin in the completion of DNA synthesis under replication stress and reveal that the Rad18/Rad5-mediated DDT pathway is linked to cohesin enrichment at sites of perturbed replication via the Snf2 family translocase Irc5.

Integrating Rio1 activities discloses its nutrient-activated network in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae kinase/adenosine triphosphatase Rio1 regulates rDNA transcription and segregation, pre-rRNA processing and small ribosomal subunit maturation. Other roles are unknown. When overexpressed, human ortholog RIOK1 drives tumor growth and metastasis. Likewise, RIOK1 promotes 40S ribosomal subunit biogenesis and has not been characterized globally. We show that Rio1 manages directly and via a series of regulators, an essential signaling network at the protein, chromatin and RNA levels. Rio1 orchestrates growth and division depending on resource availability, in parallel to the nutrient-activated Tor1 kinase. To define the Rio1 network, we identified its physical interactors, profiled its target genes/transcripts, mapped its chromatin-binding sites and integrated our data with yeast's protein-protein and protein-DNA interaction catalogs using network computation. We experimentally confirmed network components and localized Rio1 also to mitochondria and vacuoles. Via its network, Rio1 commands protein synthesis (ribosomal gene expression, assembly and activity) and turnover (26S proteasome expression), and impinges on metabolic, energy-production and cell-cycle programs. We find that Rio1 activity is conserved to humans and propose that pathological RIOK1 may fuel promiscuous transcription, ribosome production, chromosomal instability, unrestrained metabolism and proliferation; established contributors to cancer. Our study will advance the understanding of numerous processes, here revealed to depend on Rio1 activity.

Building up and breaking down: mechanisms controlling recombination during replication.

The complete and faithful duplication of the genome is an essential prerequisite for proliferating cells to maintain genome integrity. This objective is greatly challenged by DNA damage encountered during replication, which causes fork stalling and in certain cases, fork breakage. DNA damage tolerance (DDT) pathways mitigate the effects on fork stability induced by replication fork stalling by mediating damage-bypass and replication fork restart. These DDT mechanisms, largely relying on homologous recombination (HR) and specialized polymerases, can however contribute to genome rearrangements and mutagenesis. There is a profound connection between replication and recombination: recombination proteins protect replication forks from nuclease-mediated degradation of the nascent DNA strands and facilitate replication completion in cells challenged by DNA damage. Moreover, in case of fork collapse and formation of double strand breaks (DSBs), the recombination factors present or recruited to the fork facilitate HR-mediated DSB repair, which is primarily error-free. Disruption of HR is inexorably linked to genome instability, but the premature activation of HR during replication often leads to genome rearrangements. Faithful replication necessitates the downregulation of HR and disruption of active RAD51 filaments at replication forks, but upon persistent fork stalling, building up of HR is critical for the reorganization of the replication fork and for filling-in of the gaps associated with discontinuous replication induced by DNA lesions. Here we summarize and reflect on our understanding of the mechanisms that either suppress recombination or locally enhance it during replication, and the principles that underlie this regulation.

Esc2 promotes Mus81 complex-activity via its SUMO-like and DNA binding domains.

Replication across damaged DNA templates is accompanied by transient formation of sister chromatid junctions (SCJs). Cells lacking Esc2, an adaptor protein containing no known enzymatic domains, are defective in the metabolism of these SCJs. However, how Esc2 is involved in the metabolism of SCJs remains elusive. Here we show interaction between Esc2 and a structure-specific endonuclease Mus81-Mms4 (the Mus81 complex), their involvement in the metabolism of SCJs, and the effects Esc2 has on the enzymatic activity of the Mus81 complex. We found that Esc2 specifically interacts with the Mus81 complex via its SUMO-like domains, stimulates enzymatic activity of the Mus81 complex in vitro, and is involved in the Mus81 complex-dependent resolution of SCJs in vivo Collectively, our data point to the possibility that the involvement of Esc2 in the metabolism of SCJs is, in part, via modulation of the activity of the Mus81 complex.

Smc5/6 Mediated Sumoylation of the Sgs1-Top3-Rmi1 Complex Promotes Removal of Recombination Intermediates.

Timely removal of DNA recombination intermediates is critical for genome stability. The DNA helicase-topoisomerase complex, Sgs1-Top3-Rmi1 (STR), is the major pathway for processing these intermediates to generate conservative products. However, the mechanisms that promote STR-mediated functions remain to be defined. Here we show that Sgs1 binds to poly-SUMO chains and associates with the Smc5/6 SUMO E3 complex in yeast. Moreover, these interactions contribute to the sumoylation of Sgs1, Top3, and Rmi1 upon the generation of recombination structures. We show that reduced STR sumoylation leads to accumulation of recombination structures, and impaired growth in conditions when these structures arise frequently, highlighting the importance of STR sumoylation. Mechanistically, sumoylation promotes STR inter-subunit interactions and accumulation at DNA repair centers. These findings expand the roles of sumoylation and Smc5/6 in genome maintenance by demonstrating that they foster STR functions in the removal of recombination intermediates.

DNA damage tolerance by recombination: Molecular pathways and DNA structures.

Replication perturbations activate DNA damage tolerance (DDT) pathways, which are crucial to promote replication completion and to prevent fork breakage, a leading cause of genome instability. One mode of DDT uses translesion synthesis polymerases, which however can also introduce mutations. The other DDT mode involves recombination-mediated mechanisms, which are generally accurate. DDT occurs prevalently postreplicatively, but in certain situations homologous recombination is needed to restart forks. Fork reversal can function to stabilize stalled forks, but may also promote error-prone outcome when used for fork restart. Recent years have witnessed important advances in our understanding of the mechanisms and DNA structures that mediate recombination-mediated damage-bypass and highlighted principles that regulate DDT pathway choice locally and temporally. In this review we summarize the current knowledge and paradoxes on recombination-mediated DDT pathways and their workings, discuss how the intermediate DNA structures may influence genome integrity, and outline key open questions for future research.

Priming for tolerance and cohesion at replication forks.

Genome duplication is coupled with DNA damage tolerance (DDT) and chromatin structural changes. Recently we reported that mutations in Primase subunits or factors that bridge Polα/Primase with the replicative helicase, Ctf4, caused abnormal usage of DDT pathways, negatively influenced sister chromatid cohesion (SCC), and associated with increased fork reversal. (1) We also found that cohesin, which is paradigmatic for SCC, facilitates recombination-mediated DDT. However, only the recombination defects of cohesin, but not of cohesion-defective Polα/Primase/Ctf4 mutants, were rescued by artificial tethering of sister chromatids. Genetic tests and electron microscopy analysis of replication intermediates made us propose that management of single-stranded DNA forming proximal to the fork is a critical determinant of chromosome and replication fork structure, and influences DDT pathway choice. Here we discuss the implications of our findings for understanding DDT regulation and cohesion establishment during replication, and outline directions to rationalize the relationship between these chromosome metabolism processes.

The Budding Yeast Ubiquitin Protease Ubp7 Is a Novel Component Involved in S Phase Progression.

DNA damage must be repaired in an accurate and timely fashion to preserve genome stability. Cellular mechanisms preventing genome instability are crucial to human health because genome instability is considered a hallmark of cancer. Collectively referred to as the DNA damage response, conserved pathways ensure proper DNA damage recognition and repair. The function of numerous DNA damage response components is fine-tuned by posttranslational modifications, including ubiquitination. This not only involves the enzyme cascade responsible for conjugating ubiquitin to substrates but also requires enzymes that mediate directed removal of ubiquitin. Deubiquitinases remove ubiquitin from substrates to prevent degradation or to mediate signaling functions. The Saccharomyces cerevisiae deubiquitinase Ubp7 has been characterized previously as an endocytic factor. However, here we identify Ubp7 as a novel factor affecting S phase progression after hydroxyurea treatment and demonstrate an evolutionary and genetic interaction of Ubp7 with DNA damage repair pathways of homologous recombination and nucleotide excision repair. We find that deletion of UBP7 sensitizes cells to hydroxyurea and cisplatin and demonstrate that factors that stabilize replication forks are critical under these conditions. Furthermore, ubp7Δ cells exhibit an S phase progression defect upon checkpoint activation by hydroxyurea treatment. ubp7Δ mutants are epistatic to factors involved in histone maintenance and modification, and we find that a subset of Ubp7 is chromatin-associated. In summary, our results suggest that Ubp7 contributes to S phase progression by affecting the chromatin state at replication forks, and we propose histone H2B ubiquitination as a potential substrate of Ubp7.