Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae
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PROVIDER: PRJNA723670 | ENA |
REPOSITORIES: ENA
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Project description:During lagging-strand synthesis, strand-displacement synthesis by DNA polymerase delta (Pol ∂), coupled to nucleolytic cleavage of DNA flap structures, combine to produce a nick translation reaction that replaces the DNA at the 5’ end of the preceding Okazaki fragment. Previous work following depletion of DNA ligase I in Saccharomyces cerevisae suggests that DNA-bound proteins, principally nucleosomes and the transcription factors Abf1/Rap1/Reb1, pose a barrier to Pol ∂ synthesis and thereby limit the extent of nick translation in vivo. However, the extended ligase depletion required for these experiments could lead to ongoing, non-physiological nick translation. Here, we investigate nick translation by analyzing Okazaki fragments purified after transient nuclear depletion of DNA ligase I in synchronized or asynchronous S. cerevisiae cultures. We observe that, even with a short ligase depletion, Okazaki fragment termini are enriched around nucleosomes and Abf1/Reb1/Rap1 binding sites. However protracted ligase depletion leads to a global change in the location of these termini, moving them towards nucleosome dyads from a more upstream location and further enriching termini at Abf1/Reb1/Rap1 binding sites. Additionally, we observe an under-representation of DNA derived from DNA polymerase alpha – the polymerase that initiates Okazaki fragment synthesis – around the sites of Okazaki termini obtained from transient ligase depletion. Our data suggest that, while nucleosomes and transcription factors do limit strand-displacement synthesis by Pol ∂ in vivo, post-replicative nick translation can occur at unligated Okazaki fragment termini such that previous analyses represent an overestimate of the extent of nick translation occurring during normal lagging-strand synthesis.
2021-04-22 | GSE173065 | GEO
Project description: Not available
| S-EPMC8496332 | biostudies-literature
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:Although essential for epigenetic inheritance, the transfer of parental histone (H3-H4)2 tetramers that contain epigenetic modifications to replicating DNA strands is poorly understood. Here, we show that the Mcm2-Ctf4-Pol axis facilitates the transfer of parental (H3-H4)2 tetramers to lagging strands of DNA replication forks. Mutating the H3-H4 binding domain of Mcm2, a subunit of the CMG (Cdc45-MCM-GINS) replicative helicase that translocates along leading strands, impairs the transfer of parental (H3-H4)2 to lagging strands. Similar effects are observed in Ctf4 and Pol-primase mutants that disrupt the connection of the CMG helicase via Ctf4-Pol to lagging strands. Our results support a model whereby parental (H3-H4)2 displaced from nucleosomes through leading-strand DNA replication are transferred to lagging strands for nucleosome assembly via the CMG-Ctf4-Pol complex.
2018-09-24 | GSE118580 | 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 eta (pol eta) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol eta also has other cellular roles. Here, we present evidence that pol eta competes with DNA polymerases alpha and delta for the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol eta which contains a PCNA-Interacting Protein motif is required for pol eta to function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol eta and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.
2018-12-14 | GSE110241 | GEO
Project description:The division of labour between DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequence (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combining the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality, plus zones of replication initiation and termination. We confirm that transcriptional activity contributes toshapes the patterns of initiation and termination and, by separately analysing the effect of transcription ofon both both co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands in both co-directional and convergent forks is compromised by transcription. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability.
2022-11-02 | GSE189668 | 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:Cellular replicative senescence, a state of permanent cell-cycle arrest that occurs following an extended period of cell division in culture, has been linked to organismal aging, tissue repair and tumorigenesis. In this study, we comparatively investigated the global lipid profiles and mRNA content of proliferating and senescent-state BJ fibroblast cells. We found that both the expression levels of lipid-regulating genes, as well as the abundance of specific lipid families, are actively regulated. We further found that 19 polyunsaturated triacylglycerol species showed the most prominent changes during replicative senescence. We argue that diversion of polyunsaturated fatty acids to glycerolipid biosynthesis could be responsible for the accumulation of specific triacylglycerols. This, in turn, could be one of the cellular mechanisms to prevent lipotoxicity under increased oxidative stress conditions observed during replicative senescence. Collectively, our results place regulation of specific lipid species to a central role during replicative senescence.
2017-03-31 | GSE94280 | 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.
2012-02-21 | GSE33786 | GEO