Primase excites the competition of templating transcription and lagging replication to boost DNA damage
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA890279 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Head-on transcription-replication conflicts (HO-TRCs) cause R-loops and DNA damage. We realized that HO-TRCs are the natural competition for templating transcription and lagging replication in the same DNA strand (termed TemLag competition), however, the regulatory mechanisms behind are unclear. We previously identified a chloroplast-localized RNase H1 protein AtRNH1C that can remove R-loops and relax HO-TRCs for genome integrity. Through the mutagenesis screen, we identified that the chloroplast-localized primase ATH exacerbates TemLag competition in the atrnh1c mutant. A mutation in ATH weakens the binding affinity of DNA, thus slowing down DNA replication and relieving TemLag competition. Overexpression of ATH boosts TemLag competition and intensifies DNA damage. Interestingly, strand-specific DNA damage sequencing reveals that TemLag competition induces single-strand DNA damage of the competitive strand at the end of transcription. These results illustrated a potentially conserved mechanism among organisms, of which the primase antagonizes R-loop clearance machinery to exaggerate TemLag competition and genome instability.
2023-12-06 | GSE215443 | GEO
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:DNA replication fidelity is essential for maintaining genetic stability. Forks arrested at replication fork barriers can be stabilised by the intra-S phase checkpoint, subsequently being rescued by a converging fork, or resuming when the barrier is removed. However, some arrested forks cannot be stabilised and fork convergence cannot rescue in all situations. Thus, cells have developed homologous recombination-dependent mechanisms to restart persistently inactive forks. To understand the dynamics of HR-restart, we visualized in vivo replication dynamics at an S. pombe replication barrier, RTS1, using polymerase usage sequencing and model replication dynamics by Monte Carlo simulation. We confirm that HR-restarted forks synthesise both strands with Pol d and that Pol a is not used significantly on either strand: the lagging strand template remains as a gap that is filled in later. We further demonstrate that HR-restarted forks progress for >30 kb kilobases without maturing to a d/e configuration and can progress through a fork barrier that arrests canonical forks. Finally, by manipulating lagging strand resection during HR-restart by deleting pku70, we show that the leading strand initiates replication at the same position, demonstrating the stability of the 3' single strand in the context of increased resection.
2020-07-03 | GSE153731 | GEO
Project description:In response to DNA replication stress, DNA replication checkpoint is activated to maintain fork stability, a process critical for maintenance of genome stability. However, how DNA replication checkpoint regulates replication forks remain elusive. Here we show that Rad53, a highly conserved replication checkpoint kinase, functions to couple leading and lagging strand DNA synthesis. In wild type cells under HU induced replication stress, synthesis of lagging strand, which contains ssDNA gaps, is comparable to leading strand DNA. In contrast, synthesis of lagging strand is much more than leading strand, and consequently, leading template ssDNA coated with ssDNA binding protein RPA was detected in rad53-1 mutant cells, suggesting that synthesis of leading strand and lagging strand DNA is uncoupled. Mechanistically, we show that replicative helicase MCM and leading strand DNA polymerase Pole move beyond actual DNA synthesis and that an increase in dNTP pools largely suppresses the uncoupled leading and lagging strand DNA synthesis. Our studies reveal an unexpected mechanism whereby Rad53 regulates replication fork stability.
2018-02-11 | GSE89721 | GEO
Project description:We show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the chromatin repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur around nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging strand polymerase processivity impacts both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of lagging strand synthesis. Our studies represent the first high-resolution analysis of eukaryotic Okazaki fragments in vivo, and establish a mechanistic link between the fundamental processes of DNA replication and chromatin assembly. 4 samples: replicate samples of wild-type and pol32 knockout
2012-02-21 | E-GEOD-33786 | biostudies-arrayexpress
Project description:DNA polymerase delta (Pol ∂) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol ∂ is responsible for the synthesis and processing of the lagging strand; this role requires Pol ∂ to extend Okazaki fragment primers synthesized by Pol ⍺-primase, and to carry out strand-displacement synthesis coupled to nuclease cleavage during Okazaki fragment termination. Destabilizing mutations in human Pol ∂ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol ∂ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze the how the depletion of Pol ∂ impacts replication initiation and elongation in vivo in S. cerevisiae. We determine that Pol ∂ depletion leads to a dependence on checkpoint signaling and recombination-mediated repair for cellular viability. By analyzing nascent lagging-strand products, we observe both a genome-wide change in the establishment and progression of replication forks and a global defect in Pol ∂-mediated Okazaki fragment processing. Additionally, we detect significant lagging-strand synthesis by the leading-strand polymerase (Pol ɛ) in late regions of the genome when Pol ∂ is depleted.
2019-12-13 | GSE141884 | 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:How parental histone H3-H4 tetramers, the primary carrier of epigenetic modifications, are transferred to leading and lagging strands of DNA replication forks following DNA replication is an important question that remains not well understood. Here we show that DNA polymerase clamp PCNA and its partner involved in lagging strand DNA synthesis, Pol d, regulate parental histone transfer to lagging strands. Mutations at PCNA as well as at subunits of Pol d that impair the PCNA-Pol d interaction affect parental histone transfer to lagging strands, and this defect unlikely arises from their impacts on DNA synthesis. Moreover, Pol d interacts with H3-H4 in vitro. We suggest that the PCNA-Pol d complex, best known for its role in lagging strand DNA synthesis and DNA repair, couples lagging strand DNA synthesis to the transfer of the parental histones H3-H4 for the inheritance of chromatin structures following DNA replication and possibly DNA repair.
2024-05-31 | GSE203465 | GEO
Project description:Replication dynamics of recombination-dependent replication forks: leading and lagging strand synthesis are not coupled
| PRJNA643819 | ENA
Project description:The tolerance pathway allows bypassing deleterious lesions that block replicative DNA polymerases. In eukaryotes tolerance is controlled by ubiquitylation of the DNA replication factor PCNA4. Here, we show that PCNAK164-ubiquitin proteases (PCNA-DUBs) Ubp10 and Ubp12 localise to replication forks. In particular, the main PCNA-DUB, Ubp10, associates primarily to nascent lagging strands. We also found that cells deficient for PCNA deubiquitylation show both increased interaction of TLS polymerase ζ-associated Rev1 with lagging strands and Rad52-dependent template switching events. This evidence reveals a fork-coupled layer of DDT regulation allowing strand-specific pathway choices.
2019-10-29 | GSE90157 | GEO