Giacomo De Piccoli

Smc5–Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5–Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5–Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5–Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.

Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases.

The S phase checkpoint pathway preserves genome stability by protecting defective DNA replication forks, but the underlying mechanisms are still understood poorly. Previous work with budding yeast suggested that the checkpoint kinases Mec1 and Rad53 might prevent collapse of the replisome when nucleotide concentrations are limiting, thereby allowing the subsequent resumption of DNA synthesis. Here we describe a direct analysis of replisome stability in budding yeast cells lacking checkpoint kinases, together with a high-resolution view of replisome progression across the genome. Surprisingly, we find that the replisome is stably associated with DNA replication forks following replication stress in the absence of Mec1 or Rad53. A component of the replicative DNA helicase is phosphorylated within the replisome in a Mec1-dependent manner upon replication stress, and our data indicate that checkpoint kinases control replisome function rather than stability, as part of a multifaceted response that allows cells to survive defects in chromosome replication.

Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation

Mcm10 is essential for chromosome replication in eukaryotic cells and was previously thought to link the Mcm2‐7 DNA helicase at replication forks to DNA polymerase alpha. Here, we show that yeast Mcm10 interacts preferentially with the fraction of the Mcm2‐7 helicase that is loaded in an inactive form at origins of DNA replication, suggesting a role for Mcm10 during the initiation of chromosome replication, but Mcm10 is not a stable component of the replisome subsequently. Studies with budding yeast and human cells indicated that Mcm10 chaperones the catalytic subunit of polymerase alpha and preserves its stability. We used a novel degron allele to inactivate Mcm10 efficiently and this blocked the initiation of chromosome replication without causing degradation of DNA polymerase alpha. Strikingly, the other essential helicase subunits Cdc45 and GINS were still recruited to Mcm2‐7 when cells entered S‐phase without Mcm10, but origin unwinding was blocked. These findings indicate that Mcm10 is required for a novel step during activation of the Cdc45–MCM–GINS helicase at DNA replication origins.

The unnamed complex: what do we know about Smc5-Smc6?

The structural maintenance of chromosome (SMC) proteins constitute the cores of three protein complexes involved in chromosome metabolism; cohesin, condensin and the Smc5-Smc6 complex. While the roles of cohesin and condensin in sister chromatid cohesion and chromosome condensation respectively have been described, the cellular function of Smc5-Smc6 is as yet not understood, consequently the less descriptive name. The complex is involved in a variety of DNA repair pathways. It contains activities reminiscent of those described for cohesin and condensin, as well as several DNA helicases and endonucleases. It is required for sister chromatid recombination, and smc5-smc6 mutants suffer from the accumulation of unscheduled recombination intermediates. The complex contains a SUMO-ligase and potentially an ubiquitin-ligase; thus Smc5-Smc6 might presently have a dull name, but it seems destined to be recognized as a key player in the maintenance of chromosome stability. In this review we summarize our present understanding of this enigmatic protein complex.

Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication

Introduction Chromosome replication is initiated by a universal mechanism in eukaryotic cells. This mechanism entails the assembly and activation at replication origins of the DNA helicase known as CMG (Cdc45-MCM-GINS), which is essential for the progression of replication forks. The replisome is built around the CMG helicase, which associates stably with DNA replication forks until the termination of DNA synthesis. The mechanism by which CMG is disassembled was unknown until now but is likely to represent a key regulated step at the end of chromosome replication. Regulated disassembly of the CMG helicase at the end of chromosome replication in budding yeast. It is very important that the CMG helicase is not displaced from replication forks during elongation, because it cannot be reloaded. When replication terminates, however, the ubiquitin ligase SCFDia2 and the Cdc48 segregase induce disassembly of the CMG helicase, leading to dissolution of the replisome. Rationale The CMG helicase exists only at DNA replication forks but can be isolated from extracts of S-phase budding yeast cells, after digestion of chromosomal DNA. We screened for posttranslational modifications of the CMG helicase that might regulate its function. Results Here we show that the CMG helicase is ubiquitylated during the final stages of chromosome replication in Saccharomyces cerevisiae, specifically on its Mcm7 subunit. The F-box protein Dia2 is essential in vivo for ubiquitylation of CMG, and the SCFDia2 ubiquitin ligase is also required to ubiquitylate CMG in vitro on its Mcm7 subunit in extracts of S-phase yeast cells. Ubiquitylated CMG exists only transiently in vivo, as it is rapidly disassembled in a reaction that is independent of the proteasome but requires the Cdc48/p97 segregase, which associates with ubiquitylated CMG. Consistent with these data, we show that Dia2 is essential for disassembly of the CMG helicase at the end of S phase in budding yeast. Rather than causing dissolution of the active helicase, Dia2 specifically induces the disassembly of terminated CMG complexes, which suggests that the helicase undergoes a change at the end of DNA replication, predisposing it for disassembly. Conclusion Our findings indicate that the end of chromosome replication in eukaryotes is controlled in a similarly complex fashion to the much-better-characterized initiation step. Our findings indicate that replisome disassembly is driven by the regulated dissolution of the stable CMG helicase, which uses its hexameric ring of Mcm2-7 proteins to encircle the parental DNA at replication forks. Our data identify two key features of helicase disassembly in budding yeast: First, there is an essential role for the F-box protein Dia2, which drives ubiquitylation of the CMG helicase on its Mcm7 subunit. Second, the Cdc48 segregase is required to break ubiquitylated CMG into its component parts. Once separated from GINS and Cdc45, the Mcm2-7 hexamer is less stable, so that all of the subunits of the CMG helicase are lost from the newly replicated DNA. Just as the main features of helicase assembly have been conserved across evolution from yeasts to humans, we envisage that disassembly of the CMG helicase will also involve a universal mechanism in eukaryotic cells, by which Cdc48/p97 drives disassembly of ubiquitylated CMG at the end of DNA replication. How to stop after copying the genome Replication is highly regulated: Failure to copy any part of the genome or copying parts of it more than once can cause genome instability with potentially disastrous consequences. Maric et al. and Priego Moreno et al. show that the DNA replication machinery, which stably encircles DNA during the duplication process, is actively disassembled once replication is complete (see the Perspective by Bell). The protein ring encircling the DNA is covalently modified, which allows it to be opened and the whole replication complex to be removed from DNA by a special disassembly complex. Science, this issue 10.1126/science.1253596, p. 477; see also p. 418 DNA replication machinery stably encircles replicating DNA and is actively disassembled once replication is complete. [Also see Perspective by Bell] Chromosome replication is initiated by a universal mechanism in eukaryotic cells, involving the assembly and activation at replication origins of the CMG (Cdc45-MCM-GINS) DNA helicase, which is essential for the progression of replication forks. Disassembly of CMG is likely to be a key regulated step at the end of chromosome replication, but the mechanism was unknown until now. Here we show that the ubiquitin ligase known as SCFDia2 promotes ubiquitylation of CMG during the final stages of chromosome replication in Saccharomyces cerevisiae. The Cdc48/p97 segregase then associates with ubiquitylated CMG, leading rapidly to helicase disassembly. These findings indicate that the end of chromosome replication in eukaryotes is controlled in a similarly complex fashion to the much-better-characterized initiation step.

Eukaryotic Replisome Components Cooperate to Process Histones During Chromosome Replication

DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription) complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.

Surviving chromosome replication: the many roles of the S-phase checkpoint pathway

Checkpoints were originally identified as signalling pathways that delay mitosis in response to DNA damage or defects in chromosome replication, allowing time for DNA repair to occur. The ATR (ataxia- and rad-related) and ATM (ataxia-mutated) protein kinases are recruited to defective replication forks or to sites of DNA damage, and are thought to initiate the DNA damage response in all eukaryotes. In addition to delaying cell cycle progression, however, the S-phase checkpoint pathway also controls chromosome replication and DNA repair pathways in a highly complex fashion, in order to preserve genome integrity. Much of our understanding of this regulation has come from studies of yeasts, in which the best-characterized targets are the stimulation of ribonucleotide reductase activity by multiple mechanisms, and the inhibition of new initiation events at later origins of DNA replication. In addition, however, the S-phase checkpoint also plays a more enigmatic and apparently critical role in preserving the functional integrity of defective replication forks, by mechanisms that are still understood poorly. This review considers some of the key experiments that have led to our current understanding of this highly complex pathway.

The Smc5/6 complex is required for dissolution of DNA-mediated sister chromatid linkages

Mitotic chromosome segregation requires the removal of physical connections between sister chromatids. In addition to cohesin and topological entrapments, sister chromatid separation can be prevented by the presence of chromosome junctions or ongoing DNA replication. We will collectively refer to them as DNA-mediated linkages. Although this type of structures has been documented in different DNA replication and repair mutants, there is no known essential mechanism ensuring their timely removal before mitosis. Here, we show that the dissolution of these connections is an active process that requires the Smc5/6 complex, together with Mms21, its associated SUMO-ligase. Failure to remove DNA-mediated linkages causes gross chromosome missegregation in anaphase. Moreover, we show that Smc5/6 is capable to dissolve them in metaphase-arrested cells, thus restoring chromosome resolution and segregation. We propose that Smc5/6 has an essential role in the removal of DNA-mediated linkages to prevent chromosome missegregation and aneuploidy.

Transcription of ribosomal genes can cause nondisjunction.

Mitotic disjunction of the repetitive ribosomal DNA (rDNA) involves specialized segregation mechanisms dependent on the conserved phosphatase Cdc14. The reason behind this requirement is unknown. We show that rDNA segregation requires Cdc14 partly because of its physical length but most importantly because a fraction of ribosomal RNA (rRNA) genes are transcribed at very high rates. We show that cells cannot segregate rDNA without Cdc14 unless they undergo genetic rearrangements that reduce rDNA copy number. We then demonstrate that cells with normal length rDNA arrays can segregate rDNA in the absence of Cdc14 as long as rRNA genes are not transcribed. In addition, our study uncovers an unexpected role for the replication barrier protein Fob1 in rDNA segregation that is independent of Cdc14. These findings demonstrate that highly transcribed loci can cause chromosome nondisjunction.

A Conserved Motif in the C-terminal Tail of DNA Polymerase α Tethers Primase to the Eukaryotic Replisome

Background: Primase initiates DNA replication together with DNA polymerase α and forms part of the eukaryotic replisome. Results: Primase is tethered by a short motif in pol α that is functionally important. Conclusion: Tethering primase to the eukaryotic replisome is critical for normal DNA replication. Significance: Small molecule inhibitors of the primase-pol α interaction might be valuable in antitumor therapies. The DNA polymerase α-primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and pol α synthesize composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via pol α remain poorly characterized. We have identified a short conserved motif at the extreme C terminus of pol α that is critical for interaction of the yeast ortholog pol1 with primase. We show that truncation of the C-terminal residues 1452–1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid Phe1463. Conversely, a pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol α binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and pol α allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by pol α is critical for the normal action of DNA replication forks in eukaryotic cells.