Balanced act of a leading strand DNA polymerase specific domain and its exonuclease domain promotes genome-wide replication fork symmetry
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PROVIDER: PRJNA874015 | ENA |
REPOSITORIES: ENA
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Project description:During genome replication, the leading strand DNA polymerase conducts continuous synthesis over long stretches of DNA within each replicon. Processive DNA synthesis supports symmetric progression of sister replication forks and efficient genome duplication. To address the mechanisms underlying leading strand polymerase-mediated synthesis, we examine one of its conserved domains, referred to as POPS, in the budding yeast Pol2 enzyme. We provide evidence that POPS supports replication fork symmetry and efficient genome replication via balancing the function of the Pol2 exonuclease domain. We found that the defective growth, slow S phase progression, and impaired genome synthesis associated with a POPS mutation were rescued by abolishing the Pol2 exonuclease activity. The suppressive effects further extended to the increased DNA re-arrangements in the POPS mutant and its negative genetic interactions with mutants of other genome maintenance factors. Significantly, our single molecule replicon-seq data demonstrate that the POPS mutant exhibited genome-wide replication fork asymmetry, and this defect was improved by eliminating the Pol2 exonuclease activity, thus providing a basis for its rescuing of a range of POPS mutant phenotypes. Collectively, these data suggest a model in which balanced activity between a unique Pol2 catalytic domain, and its exonuclease domain facilitates replication fork symmetry and genome maintenance.
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Project description:DNA polymerase epsilon (Pole) carries out leading strand synthesis with high fidelity owing to its exonuclease activity. Pole polymerase and exonuclease activities are in balance, due to partitioning of nascent strands between catalytic sites, so that net end resection occurs when synthesis is impaired. Stalling of chromosomal DNA synthesis activates replication checkpoint kinases, required to preserve the functional integrity of replication forks. We found that Pole is phosphorylated in a Rad53CHK1-dependent manner upon fork stalling, likely to limit Pole-driven nascent strand resection that causes replication fork collapse. In stress conditions Pole phosphorylation occurs on serine 430 of the Pol2 catalytic subunit. A S430 phosphomimic limits strand partitioning and exonucleolytic processivity, while non-phosphorylatable Pol2-S430A bypasses checkpoint regulation causing stalled fork resection and collapse. We propose that checkpoint kinases switch Pole to an exonuclease-safe mode by curbing active site partitioning thus preventing nascent strand resection and stabilizing stalled replication forks.
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Project description:Checkpoint-mediated DNA polymerase e exonuclease activity curbing counteracts resection-driven fork collapse
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Project description:DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600 bp region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M/G1 transition. top1 top2 double mutants exhibit fork block and processing, and phosphorylation of Rad53 and γH2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation and chromosome breakage during segregation. Keywords: ChIP-chip analysis
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Project description:Exonuclease domain-containing 1 enhances MIWI2 piRNA biogenesis via its interaction with TDRD12
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Project description:PIWI proteins and their associated small RNAs called PIWI-interacting RNAs (piRNAs) restrict transposon activity in animal gonads to ensure fertility. Distinct biogenesis pathways load piRNAs into the PIWI proteins MILI and MIWI2 in the mouse male embryonic germline. While most of MILI piRNAs derive via a slicer-independent pathway, a MILI slicer endonuclease-initiated pathway loads nuclear MIWI2 with a series of phased piRNAs. Tudor domain-containing 12 (TDRD12) and its interaction partner Exonuclease domain-containing 1 (EXD1) are required for loading MIWI2, but only Tdrd12 is essential for fertility, leaving us with no explanation for the physiological role of Exd1. Using an artificial piRNA precursor, we demonstrate that MILI-triggered piRNA biogenesis is greatly reduced in the Exd1 mutant. The situation deteriorates in the sensitized Exd1 mutant (Exd1-/-; Tdrd12+/-), where diminished MIWI2 piRNA levels de-repress LINE1 retrotransposons, causing infertility. Thus, EXD1 enhances slicing-triggered MIWI2 piRNA biogenesis via a functional interaction with TDRD12.
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Project description:Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3′–5′ domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.
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Project description:Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR’s SNF2 domain, we identified a physical interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in an SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a direct regulator of replication fork progression suggesting developmental regulation of Rif1 activity.
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Project description:Chronic myeloid leukemia (CML), caused by BCR::ABL1 fusion gene, is known to regulate disease progression by altering the expression of genes. However, the molecular mechanisms underlying these changes are largely unknown. In this study, we identified RNA Exonuclease 5 (REXO5) as a novel gene with elevated mRNA expression levels in chronic myeloid leukemia (CML) patients. Additionally, using the REXO5 knockout (KO) K562 cell lines, we revealed a novel role for REXO5 in the DNA damage response (DDR). Compared to wild-type (WT) cells, REXO5 KO cells showed an accumulation of R-loops and increased DNA damage. We demonstrated that REXO5 translocates to sites of DNA damage through its RNA recognition motif (RRM) and selectively binds to R loops. Interestingly, we identified that REXO5 regulates R-loop levels by degrading mRNA within R-loop using its exonuclease domain. REXO5 KO showed ATR-CHK1 activation. Collectively, we demonstrated that REXO5 plays a key role in the physiological control of R-loops using its exonuclease domain. These findings may provide novel insights into how REXO5 expression contribute to CML pathogenesis.
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