DNA polymerase Δ synthesizes both strands during break-induced replication
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA551884 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9621950_1.fastq.gz | Fastqsanger.gz | |||
| SRR9621950_2.fastq.gz | Fastqsanger.gz | |||
| SRR9621960_1.fastq.gz | Fastqsanger.gz | |||
| SRR9621960_2.fastq.gz | Fastqsanger.gz | |||
| SRR9621970_1.fastq.gz | Fastqsanger.gz |
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Project description:We applied our ribonucleotide mapping technique HydEn-seq to study DNA polymerase usage during break-induced replication (BIR) in the budding yeast Saccharomyces cerevisiae.
2019-07-01 | GSE133558 | GEO
Project description:Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol ?) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized. By using alleles of the replicative DNA polymerases that are permissive for ribonucleotide incorporation, thus generating a signature of their action in the genome that can be identified by hydrolytic end sequencing, we show that Pol ? replicates both the invading and the complementary strand during BIR. In support of this conclusion, we show that depletion of Pol ? from cells reduces BIR, whereas depletion of Pol ? has no effect.
| S-EPMC6862718 | biostudies-literature
Project description:Chromatin replication is intricately intertwined with DNA replication, and the recycling of parental histones is essential for epigenetic inheritance. The transfer of parental histones to both the DNA replication leading and lagging strands involves two distinct pathways: the leading strand utilizes DNA polymerase ε subunits Dpb3/Dpb4, while the lagging strand is facilitated by the MCM helicase subunit Mcm2. However, the process by which Mcm2, moving along the leading strand, facilitates the transfer of parental histones to the lagging strand remains unclear. Our study reveals that the deletion of Pol32, a non-essential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the replication leading strand. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted by the mutation of Mcm2's histone H3-H4 binding domain. In conclusion, our findings identify the DNA polymerase delta subunit Pol32 as a novel key histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.
2024-04-24 | GSE252049 | GEO
Project description:Genetic studies with S. cerevisiae Polδ (pol3-L612M) and Polε (pol2-M644G) mutant alleles, each of which display a higher rate for the generation of a specific mismatch, have led to the conclusion that Polε is the primary leading strand replicase and that Polδ is restricted to replicating the lagging strand template. Contrary to this widely accepted view, here we show that Polδ plays a major role in the replication of both DNA strands, and that the paucity of pol3-L612M-generated errors on the leading strand results from their more proficient removal. Thus, the apparent lack of Polδ contribution to leading strand replication is due to differential mismatch removal rather than differential mismatch generation. Altogether, our genetic studies with Pol3 and Pol2 mutator alleles support the conclusion that Polδ, and not Polε, is the major DNA polymerase for carrying out both leading and lagging DNA synthesis.
| S-EPMC4517859 | biostudies-literature
Project description:To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase δ (Pol δ). Although pol2-16 mutants survive, their spore colonies are very tiny, with increased doubling time, larger than normal cells, aberrant nuclei, and rapid suppressor mutation accumulation. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack Pol δ proofreading (pol2-4), consistent with the idea that Pol δ is the major leading strand replicase. Ribonucleotides are also incorporated into the pol2-16 genome in patterns consistent with leading strand replication by Pol δ when Pol δ is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol δ contributes to initiation of leading-strand replication. We describe two possible models.
2018-02-27 | GSE101698 | GEO
Project description:During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitution, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Pole) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Pold). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.
2023-08-08 | GSE223782 | GEO
Project description:To maintain genetic stability, DNA must be replicated only once per cell cycle, and replication must be completed even when individual replication forks are inactivated. Because fork inactivation is common, passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination-dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via homologous recombination-restarted replication (HoRReR) ensures cell survival, but at a cost. One such cost is increased mutagenesis because HoRReR is more error prone than canonical replication. This increased error rate implies the HoRReR mechanism is distinct from that of a canonical fork. Here we demonstrate, in Schizosaccharomyces pombe, that a DNA sequence duplicated by HoRReR during S phase is replicated semiconservatively, but both the leading and lagging strands are synthesized by DNA polymerase ?.
| S-EPMC4655445 | biostudies-literature
Project description:Transcription and replication conflict (TRC) are one of the main driving forces for genome instability. Yet, TRC rarely been discussed without the context of DNA:RNA entanglement, rending the role of transcription in other TRC unclear . In neural stem and progenitor cells, genes encode protein regulating neuron adhesion are hotspots for recurrent DNA break clusters (RDC). While RDC-containing genes are all actively transcribed, most RDC lack DNA:RNA entanglement. We demonstrated that, through controlled gain and loss of function genetic approaches, transcription activity is essential while not sufficient to induce RDC formation. In combination of a deep neural network and single-nucleotide resolution DNA break mapping approaches, we found RDC break densities mirror the replication fork dynamics. We demonstrated that, for the first time that, head-on TRC results in higher DNA break density than its co-direction counterparts. In summary, our results revealed that transcription has a higher-level regulatory role that has to be coordinated with DNA replication.
2024-03-18 | GSE233842 | GEO
Project description:Recent studies have implicated DNA polymerases θ (Pol θ) and β (Pol β) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.
| S-EPMC7959522 | biostudies-literature
Project description:Twenty-one pheromone-induced genes were selected from the literature (Zhao, Daniels et al. 2005 was the major source) as the reference set for assessing the pheromone response of CAI4 (Wild-type), cpp1Δ/Δ, cek1Δ/Δ, cek2Δ/Δ, cpp1Δ/Δ cek1Δ/Δ, cpp1Δ/Δ cek2Δ/Δ and cek1Δ/Δ cek2Δ/Δ strains.Our aim was to check whether or not these 21 pheromone-induced genes are up-regulated in response to pheromone in each mutant strain.
2019-01-26 | GSE125636 | GEO