RESUMEN
DNA replication is a fundamental process for cell proliferation, governed by intricate mechanisms involving leading and lagging strand synthesis. In eukaryotes, canonical DNA replication occurs during the S phase of the cell cycle, facilitated by various components of the replicative machinery at sites known as replication origins. Leading and lagging strands exhibit distinct replication dynamics, with leading strand replication being relatively straightforward compared to the complex synthesis of lagging strands involving Okazaki fragment maturation. Central to DNA synthesis are DNA polymerases, with Polα, Polε, and Polδ playing pivotal roles, each specializing in specific tasks during replication. Notably, leading and lagging strands are replicated by different polymerases, contributing to the division of labor in DNA replication. Understanding the enzymology of asymmetric DNA replication has been challenging, with methods relying on ribonucleotide incorporation and next-generation sequencing techniques offering comprehensive insights. These methodologies, such as HydEn-seq, PU-seq, ribose-seq, and emRiboSeq, offer insights into polymerase activity and strand synthesis, aiding in understanding DNA replication dynamics. Recent advancements include novel conditional mutants for ribonucleotide excision repair, enzymatic cleavage alternatives, and unified pipelines for data analysis. Further developments in adapting techniques to different organisms, studying non-canonical polymerases, and exploring new sequencing platforms hold promise for expanding our understanding of DNA replication dynamics. Integrating strand-specific information into single-cell studies could offer novel insights into enzymology, opening avenues for future research and applications in repair and replication biology.
RESUMEN
The S-phase checkpoint is involved in coupling DNA unwinding with nascent strand synthesis and is critical to maintain replication fork stability in conditions of replicative stress. However, its role in the specific regulation of leading and lagging strands at stalled forks is unclear. By conditionally depleting RNaseH2 and analyzing polymerase usage genome-wide, we examine the enzymology of DNA replication during a single S-phase in the presence of replicative stress and show that there is a differential regulation of lagging and leading strands. In checkpoint proficient cells, lagging strand replication is down-regulated through an Elg1-dependent mechanism. Nevertheless, when checkpoint function is impaired we observe a defect specifically at the leading strand, which was partially dependent on Exo1 activity. Further, our genome-wide mapping of DNA single-strand breaks reveals that strand discontinuities highly accumulate at the leading strand in HU-treated cells, whose dynamics are affected by checkpoint function and Exo1 activity. Our data reveal an unexpected role of Exo1 at the leading strand and support a model of fork stabilization through prevention of unrestrained Exo1-dependent resection of leading strand-associated nicks after fork stalling.
Asunto(s)
Roturas del ADN de Cadena Simple , Replicación del ADN , Exodesoxirribonucleasas , Puntos de Control de la Fase S del Ciclo Celular , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ribonucleasa H/metabolismo , Ribonucleasa H/genética , Fase S/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMEN
The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1-2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants.