Sen1 Is Recruited to Replication Forks via Ctf4 and Mrc1 and Promotes Genome Stability.

DNA replication and RNA transcription compete for the same substrate during S phase. Cells have evolved several mechanisms to minimize such conflicts. Here, we identify the mechanism by which the transcription termination helicase Sen1 associates with replisomes. We show that the N terminus of Sen1 is both sufficient and necessary for replisome association and that it binds to the replisome via the components Ctf4 and Mrc1. We generated a separation of function mutant, sen1-3, which abolishes replisome binding without affecting transcription termination. We observe that the sen1-3 mutants show increased genome instability and recombination levels. Moreover, sen1-3 is synthetically defective with mutations in genes involved in RNA metabolism and the S phase checkpoint. RNH1 overexpression suppresses defects in the former, but not the latter. These findings illustrate how Sen1 plays a key function at replication forks during DNA replication to promote fork progression and chromosome stability.

The S phase checkpoint promotes the Smc5/6 complex dependent SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε.

Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding ability.

A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1.

Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the 'alternative clamp loader' known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved 'Pol ϵ binding module' in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint.

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

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 SCF(Dia2) 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.

Dpb2 integrates the leading-strand DNA polymerase into the eukaryotic replisome.

BACKGROUND: The eukaryotic replisome is a critical determinant of genome integrity with a complex structure that remains poorly characterized. A central unresolved issue is how the Cdc45-MCM-GINS helicase is linked to DNA polymerase epsilon, which synthesizes the leading strand at replication forks and is an important focus of regulation. RESULTS: Here, we use budding yeast to show that a conserved amino-terminal domain of the Dpb2 subunit of Pol ε (Dpb2NT) interacts with the Psf1 component of GINS, via the unique "B domain" of the latter that is dispensable for assembly of the GINS complex but is essential for replication initiation. We show that Dpb2NT is required during initiation for assembly of the Cdc45-MCM-GINS helicase. Moreover, overexpressed Dpb2NT is sufficient to support assembly of the Cdc45-MCM-GINS helicase during initiation, upon depletion of endogenous Dpb2. This produces a replisome that lacks DNA polymerase epsilon, and although cells are viable, they grow extremely poorly. Finally, we use a novel in vitro assay to show that Dpb2NT is essential for Pol ε to interact with the replisome after initiation. CONCLUSIONS: These findings indicate that the association of Dpb2 with the B domain of Psf1 plays two critical roles during chromosome replication in budding yeast. First, it is required for initiation, because it facilitates the incorporation of GINS into the Cdc45-MCM-GINS helicase at nascent forks. Second, it plays an equally important role after initiation, because it links the leading strand DNA polymerase to the Cdc45-MCM-GINS helicase within the replisome.

A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome.

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 Phe(1463). 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.

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.

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.

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.

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.

Can eukaryotic cells monitor the presence of unreplicated DNA?

Completion of DNA replication before mitosis is essential for genome stability and cell viability. Cellular controls called checkpoints act as surveillance mechanisms capable of detecting errors and blocking cell cycle progression to allow time for those errors to be corrected. An important question in the cell cycle field is whether eukaryotic cells possess mechanisms that monitor ongoing DNA replication and make sure that all chromosomes are fully replicated before entering mitosis, that is whether a replication-completion checkpoint exists. From recent studies with smc5-smc6 mutants it appears that yeast cells can enter anaphase without noticing that replication in the ribosomal DNA array was unfinished. smc5-smc6 mutants are proficient in all known cellular checkpoints, namely the S phase checkpoint, DNA-damage checkpoint, and spindle checkpoint, thus suggesting that none of these checkpoints can monitor the presence of unreplicated segments or the unhindered progression of forks in rDNA. Therefore, these results strongly suggest that normal yeast cells do not contain a DNA replication-completion checkpoint.

The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus.

Homologous recombination (HR) is crucial for maintaining genome integrity by repairing DNA double-strand breaks (DSBs) and rescuing collapsed replication forks. In contrast, uncontrolled HR can lead to chromosome translocations, loss of heterozygosity, and deletion of repetitive sequences. Controlled HR is particularly important for the preservation of repetitive sequences of the ribosomal gene (rDNA) cluster. Here we show that recombinational repair of a DSB in rDNA in Saccharomyces cerevisiae involves the transient relocalization of the lesion to associate with the recombination machinery at an extranucleolar site. The nucleolar exclusion of Rad52 recombination foci entails Mre11 and Smc5-Smc6 complexes and depends on Rad52 SUMO (small ubiquitin-related modifier) modification. Remarkably, mutations that abrogate these activities result in the formation of Rad52 foci within the nucleolus and cause rDNA hyperrecombination and the excision of extrachromosomal rDNA circles. Our study also suggests a key role of sumoylation for nucleolar dynamics, perhaps in the compartmentalization of nuclear activities.

Anaphase onset before complete DNA replication with intact checkpoint responses.

Cellular checkpoints prevent mitosis in the presence of stalled replication forks. Whether checkpoints also ensure the completion of DNA replication before mitosis is unknown. Here, we show that in yeast smc5-smc6 mutants, which are related to cohesin and condensin, replication is delayed, most significantly at natural replication-impeding loci like the ribosomal DNA gene cluster. In the absence of Smc5-Smc6, chromosome nondisjunction occurs as a consequence of mitotic entry with unfinished replication despite intact checkpoint responses. Eliminating processes that obstruct replication fork progression restores the temporal uncoupling between replication and segregation in smc5-smc6 mutants. We propose that the completion of replication is not under the surveillance of known checkpoints.

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.

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.