Lagging strand detection with SDL-TOPO protocol
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ABSTRACT:
PROVIDER: PRJNA1208944 | ENA |
REPOSITORIES: ENA
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Project description:SDL-TOPO protocol was applied for the detection of lagging-strand in replicating budding yeast strain. Genomic DNA was extracted from cdc9 mutant strain, and served for library preparation with SDL-TOPO. To see the sensitivity of SDL-TOPO for the detection of lagging strand, various amounts of genomic DNA were used for the library preparation: 75 ng, 25 ng, 7.5 ng, 2.5 ng, 750 pg, 250 pg, 75 pg, and 25 pg. The prepared libraries were sequenced on Illumina iSeq100 with paired-end mode of 150x2. Only read 1 was served for mapping with bowtie2 against reference sequence sacSer3. The mapped reads were separated strand specifically, and read depths were visualized. The strand-biased mapping patterens were observed for libraries prepared from each amount of input DNA.
2025-01-17 | GSE286342 | 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: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 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: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: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
Project description:DNA polymerase η contributes to lagging strand synthesis
| PRJNA433239 | ENA
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:Ribonucleotides are frequently incorporated into DNA and can be used as a marker of DNA replication enzymology. To investigate on a genome-wide scale, how E. coli pol V accesses undamaged chromosomal DNA during the SOS response, we mapped the location of ribonucleotides incorporated by steric gate variants of pol V across the entire E. coli genome. To do so, we used strains that are deficient in ribonucleotide excision repair (DrnhB), constitutively express all SOS-regulated genes [lexA(Def)] and constitutively “activated” RecA* (recA730). The strains also harbor two steric gate variants of E. coli pol V (Y11A or F10L), or a homolog of pol V, (pol VR391-Y13A). Ribonucleotides are frequently incorporated by the pol V-Y11A and pol VR391-Y13A variants, with a preference to the lagging strand. In contrast, the pol V-F10L variant incorporates less ribonucleotides and no strand preference was observed. Sharp transitions in strand specificity are observed at replication origin (oriC), while a gradient is observed at the termination region. To activate RecA* in a recA+ strain, we treated the strains with ciprofloxacin and genome-wide mapped the location of the incorporated ribonucleotides. Again, the polV-Y11A steric gate variant, exhibited a lagging strand preference. Our data is consistent with a specific role for pol V in lagging strand DNA synthesis across the entire E. coli genome during the SOS response.
2021-03-22 | GSE158570 | GEO