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A new mechanism of chromosomal rearrangement is identified through the observation that broken or collapsed DNA replication forks restarted by homologous recombination have a high propensity for U-turns at short inverted repeats; the error-prone nature of this mechanism is suggested to contribute to gross chromosomal rearrangements and copy-number variations present in cancer and other genomic disorders. Paused replication forks a source of gene abnormality Stress that causes broken or collapsed DNA replication forks can induce copy-number variations (CNVs) and gross chromosomal rearrangements (GCRs), both of which are commonly found in cancer cells. Antony Carr and colleagues find that a collapsed fork that has been restarted by homologous recombination is more error prone than a fork that initiates at origins of replication. These restarted forks frequently perform U-turns at short inverted repeats, a possible cause of CNVs and GCRs. The authors suggest that the error-prone nature of restarted forks could be a source of cancer-causing gene abnormalities and spontaneous genetic disorders in the absence of a double-strand break. Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders 1 and are a feature of cancer 2 . During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance 3 . When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S-phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination or microhomology-primed re-initiation 4 , 5 . Here we ascertain the consequences of replication with a fork restarted by homologous recombination in fission yeast. We identify a new mechanism of chromosomal rearrangement through the observation that recombination-restarted forks have a considerably high propensity to execute a U-turn at small inverted repeats (up to 1 in 40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer, and to non-recurrent CNVs in genomic disorders.

The Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin and Smc5/6 are involved in the organization of higher-order chromosome structure—which is essential for accurate chromosome duplication and segregation. Each complex is scaffolded by a specific SMC protein dimer (heterodimer in eukaryotes) held together via their hinge domains. Here we show that the Smc5/6-hinge, like those of cohesin and condensin, also forms a toroidal structure but with distinctive subunit interfaces absent from the other SMC complexes; an unusual ‘molecular latch’ and a functional ‘hub’. Defined mutations in these interfaces cause severe phenotypic effects with sensitivity to DNA-damaging agents in fission yeast and reduced viability in human cells. We show that the Smc5/6-hinge complex binds preferentially to ssDNA and that this interaction is affected by both ‘latch’ and ‘hub’ mutations, suggesting a key role for these unique features in controlling DNA association by the Smc5/6 complex. Structural Maintenance of Chromosomes (SMC) complexes maintain genome integrity by regulating the segregation of chromosomes. Here, Alt et al . describe the structure of the heterodimeric Smc5/6-hinge from fission yeast and define functional features critical for Smc5/6’s cellular function.

The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits, we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

Thymidine kinase converts 5-fluorodeoxyuridine to 5-fluorodeoxyuridine monophosphate, which causes disruption of deoxynucleotide triphosphate ratios. The fission yeast Schizosaccharomyces pombe does not express endogenous thymidine kinase but 5-fluorodeoxyuridine inhibits growth when exogenous thymidine kinase is expressed. Unexpectedly, we found that 5-fluorodeoxyuridine causes S phase arrest even without thymidine kinase expression. DNA damage checkpoint proteins such as the 9-1-1 complex were required for viability in the presence of 5-fluorodeoxyuridine. We also found that strains with circular chromosomes, due to loss of pot1+, which have higher levels of replication stress, were more sensitive to loss of the 9-1-1 complex in the presence of 5-fluorodeoxyuridine. Thus, our results suggest that strains carrying circular chromosomes exhibit a greater dependence on DNA damage checkpoints to ensure viability in the presence of 5-fluorodeoxyuridine compared to stains that have linear chromosomes.

Structural Maintenance of Chromosomes (SMC) complexes are important for many aspects of the chromosomal organization. Unlike cohesin and condensin, the SMC5/6 complex contains a variant RING domain carried by its Nse1 subunit. RING domains are characteristic for ubiquitin ligases, and human NSE1 has been shown to possess ubiquitin-ligase activity in vitro. However, other studies were unable to show such activity. Here, we confirm Nse1 ubiquitin-ligase activity using purified Schizosaccharomyces pombe proteins. We demonstrate that the Nse1 ligase activity is stimulated by Nse3 and Nse4. We show that Nse1 specifically utilizes Ubc13/Mms2 E2 enzyme and interacts directly with ubiquitin. We identify the Nse1 mutation (R188E) that specifically disrupts its E3 activity and demonstrate that the Nse1-dependent ubiquitination is particularly important under replication stress. Moreover, we determine Nse4 (lysine K181) as the first known SMC5/6-associated Nse1 substrate. Interestingly, abolition of Nse4 modification at K181 leads to suppression of DNA-damage sensitivity of other SMC5/6 mutants. Altogether, this study brings new evidence for Nse1 ubiquitin ligase activity, significantly advancing our understanding of this enigmatic SMC5/6 function.

In the original publication [...]

The Smc5/6 structural maintenance of chromosomes complex is required for efficient homologous recombination (HR). Defects in Smc5/6 result in chromosome mis‐segregation and fragmentation. By characterising two Schizosaccharomyces pombe smc6 mutants, we define two separate functions for Smc5/6 in HR. The first represents the previously described defect in processing recombination‐dependent DNA intermediates when replication forks collapse, which leads to increased rDNA recombination. The second novel function defines Smc5/6 as a positive regulator of recombination in the rDNA and correlates mechanistically with a requirement to load RPA and Rad52 onto chromatin genome‐wide when replication forks are stably stalled by nucleotide depletion. Rad52 is required for all HR repair, but Rad52 loading in response to replication fork stalling is unexpected and does not correlate with damage‐induced foci. We propose that Smc5/6 is required to maintain stalled forks in a stable recombination‐competent conformation primed for replication restart.

The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the \"OFF-state\". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down Purg1 \"OFF-levels\" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining Purg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair.

DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2. However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2 -mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.

New analyses in S. pombe show that both the leading and lagging strands are replicated by DNA polymerase δ when stalled replication forks use homologous recombination to restart semiconservative DNA synthesis. To maintain genetic stability, DNA must be replicated only once per cell cycle, and replication must be completed even when individual replication forks are inactivated. Because fork inactivation is common, passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination–dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via homologous recombination–restarted replication (HoRReR) ensures cell survival, but at a cost. One such cost is increased mutagenesis because HoRReR is more error prone than canonical replication. This increased error rate implies the HoRReR mechanism is distinct from that of a canonical fork. Here we demonstrate, in Schizosaccharomyces pombe , that a DNA sequence duplicated by HoRReR during S phase is replicated semiconservatively, but both the leading and lagging strands are synthesized by DNA polymerase δ.