Armelle Lengronne

Cohesin relocation from sites of chromosomal loading to places of convergent transcription.

Sister chromatids, the products of eukaryotic DNA replication, are held together by the chromosomal cohesin complex after their synthesis. This allows the spindle in mitosis to recognize pairs of replication products for segregation into opposite directions. Cohesin forms large protein rings that may bind DNA strands by encircling them, but the characterization of cohesin binding to chromosomes in vivo has remained vague. We have performed high resolution analysis of cohesin association along budding yeast chromosomes III–VI. Cohesin localizes almost exclusively between genes that are transcribed in converging directions. We find that active transcription positions cohesin at these sites, not the underlying DNA sequence. Cohesin is initially loaded onto chromosomes at separate places, marked by the Scc2/Scc4 cohesin loading complex, from where it appears to slide to its more permanent locations. But even after sister chromatid cohesion is established, changes in transcription lead to repositioning of cohesin. Thus the sites of cohesin binding and therefore probably sister chromatid cohesion, a key architectural feature of mitotic chromosomes, display surprising flexibility. Cohesin localization to places of convergent transcription is conserved in fission yeast, suggesting that it is a common feature of eukaryotic chromosomes.

The Yeast CDK Inhibitor Sic1 Prevents Genomic Instability by Promoting Replication Origin Licensing in Late G1

G(1) cell cycle regulators are often mutated in cancer, but how this causes genomic instability is unclear. Here we show that yeast lacking the CDK inhibitor Sic1 initiate DNA replication from fewer origins, have an extended S phase, and inefficiently separate sister chromatids during anaphase. This leads to double-strand breaks (DSBs) in a fraction of sic1 cells as evidenced by the accumulation of Ddc1 foci and a 575-fold increase in gross chromosomal rearrangements. Both S and M phase defects are rescued by delaying S-CDK activation, indicating that Sic1 promotes origin licensing in late G(1) by preventing the untimely activation of CDKs. We propose that precocious CDK activation causes genomic instability by altering the dynamics of S phase, which then hinders normal chromosome segregation.

dNTP pools determine fork progression and origin usage under replication stress

Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow‐replication mode within minutes after S‐phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.

Analysis of replication profiles reveals key role of RFC-Ctf18 in yeast replication stress response

Maintenance of genome integrity relies on surveillance mechanisms that detect and signal arrested replication forks. Although evidence from budding yeast indicates that the DNA replication checkpoint (DRC) is primarily activated by single-stranded DNA (ssDNA), studies in higher eukaryotes have implicated primer ends in this process. To identify factors that signal primed ssDNA in Saccharomyces cerevisiae, we have screened a collection of checkpoint mutants for their ability to activate the DRC, using the repression of late origins as readout for checkpoint activity. This quantitative analysis reveals that neither RFCRad24 and the 9-1-1 clamp nor the alternative clamp loader RFCElg1 is required to signal paused forks. In contrast, we found that RFCCtf18 is essential for the Mrc1-dependent activation of Rad53 and for the maintenance of paused forks. These data identify RFCCtf18 as a key DRC mediator, potentially bridging Mrc1 and primed ssDNA to signal paused forks.

Ctf4 Links DNA Replication with Sister Chromatid Cohesion Establishment by Recruiting the Chl1 Helicase to the Replisome

Summary DNA replication during S phase is accompanied by establishment of sister chromatid cohesion to ensure faithful chromosome segregation. The Eco1 acetyltransferase, helped by factors including Ctf4 and Chl1, concomitantly acetylates the chromosomal cohesin complex to stabilize its cohesive links. Here we show that Ctf4 recruits the Chl1 helicase to the replisome via a conserved interaction motif that Chl1 shares with GINS and polymerase α. We visualize recruitment by EM analysis of a reconstituted Chl1-Ctf4-GINS assembly. The Chl1 helicase facilitates replication fork progression under conditions of nucleotide depletion, partly independently of Ctf4 interaction. Conversely, Ctf4 interaction, but not helicase activity, is required for Chl1’s role in sister chromatid cohesion. A physical interaction between Chl1 and the cohesin complex during S phase suggests that Chl1 contacts cohesin to facilitate its acetylation. Our results reveal how Ctf4 forms a replisomal interaction hub that coordinates replication fork progression and sister chromatid cohesion establishment.

Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression

Significance During each cell-division cycle, eukaryotic cells initiate DNA synthesis from multiple replication origins on chromosomes to duplicate the entire genome once and only once. Spatial and temporal control of initiation and subsequent DNA synthesis at replication forks is important for maintaining genome integrity. Here we present a comprehensive analysis of patterns of origin activation, replication fork progression, and checkpoint responses in cells under replication stress. Our studies showed that a domain intrinsic to the replicative helicase, which unwinds DNA during replication, integrates multiple kinase-signaling pathways to control various aspects of the genome duplication process. Our work suggests a mechanism by which eukaryotic cells modulate the pattern of replication in response to environmental conditions through the replicative helicase. Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions.

Essential Roles of the Smc5/6 Complex in Replication through Natural Pausing Sites and Endogenous DNA Damage Tolerance

Summary The essential functions of the conserved Smc5/6 complex remain elusive. To uncover its roles in genome maintenance, we established Saccharomyces cerevisiae cell-cycle-regulated alleles that enable restriction of Smc5/6 components to S or G2/M. Unexpectedly, the essential functions of Smc5/6 segregated fully and selectively to G2/M. Genetic screens that became possible with generated alleles identified processes that crucially rely on Smc5/6 specifically in G2/M: metabolism of DNA recombination structures triggered by endogenous replication stress, and replication through natural pausing sites located in late-replicating regions. In the first process, Smc5/6 modulates remodeling of recombination intermediates, cooperating with dissolution activities. In the second, Smc5/6 prevents chromosome fragility and toxic recombination instigated by prolonged pausing and the fork protection complex, Tof1-Csm3. Our results thus dissect Smc5/6 essential roles and reveal that combined defects in DNA damage tolerance and pausing site-replication cause recombination-mediated DNA lesions, which we propose to drive developmental and cancer-prone disorders.

Closing the MCM cycle at replication termination sites

The initiation of eukaryotic DNA replication is a highly regulated process conserved from yeast to human. The past decade has seen significant advances in understanding how the CMG (Cdc45‐MCM‐GINS) replicative helicase is loaded onto DNA. However, very little was known on how this complex is removed from chromatin at the end of S phase. Two papers in a recent issue of Science show that in yeast and in Xenopus, the CMG complex is unloaded at replication termination sites by an active mechanism involving the polyubiquitylation of Mcm7.

The replication timing program in the hands of two HDACs

In eukaryotes, duplication of genomic information depends on the sequential activation of multiple replication origins distributed along the chromosomes. Replication origins differ in initiation time, chromatin structure and three-dimensional position in the nucleus. Recently, we have performed a systematic analysis of the role of histone deacetylases (HDACs) in the regulation of origin activity in budding yeast. We have found that the epigenetic regulation of repetitive sequences is a key determinant of the DNA replication program. Indeed, our study revealed that two histone deacetylases, Rpd3 and Sir2, have opposite effects on the replication timing program. Rpd3 delays initiation at late origins, whereas Sir2 promotes efficient activation of early origins. Remarkably, we also found that Rpd3 and Sir2 regulate initiation at ~200 replication origins located within the ribosomal DNA (rDNA) array. We propose that this epigenetic regulation of repetitive origins controls the replication timing program by modulating the availability of limiting initiation factors.

Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage

The S‐phase checkpoint maintains the integrity of the genome in response to DNA replication stress. In budding yeast, this pathway is initiated by Mec1 and is amplified through the activation of Rad53 by two checkpoint mediators: Mrc1 promotes Rad53 activation at stalled forks, and Rad9 is a general mediator of the DNA damage response. Here, we have investigated the interplay between Mrc1 and Rad9 in response to DNA damage and found that they control DNA replication through two distinct but complementary mechanisms. Mrc1 rapidly activates Rad53 at stalled forks and represses late‐firing origins but is unable to maintain this repression over time. Rad9 takes over Mrc1 to maintain a continuous checkpoint signaling. Importantly, the Rad9‐mediated activation of Rad53 slows down fork progression, supporting the view that the S‐phase checkpoint controls both the initiation and the elongation of DNA replication in response to DNA damage. Together, these data indicate that Mrc1 and Rad9 play distinct functions that are important to ensure an optimal completion of S phase under replication stress conditions.