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Next-generation sequencing of Okazaki fragments extracted from Saccharomyces cerevisiae

ABSTRACT: We investigated the genome-wide distribution of Okazaki fragments in the commonly used laboratory Saccharomyces cerevisiae strain S288C to study the DNA replication model adopted by the budding yeast. The method based upon lambda exonuclease digestion for purification of RNA-primed replication intermediates was first improved to be suitable for the purification of Okazaki fragments. Then, we used this improved method to purify Okazaki fragments from S288C yeast cells, followed by Illumina sequencing. We found that the expected asymmetric distribution of Okazaki fragments around confirmed replication origins, which was derived from the semi-discontinuous DNA replication model, was not observed on S. cerevisiae chromosomes. Even around two highly efficient replication origins, ARS522 and ARS416, the ratios of Okazaki fragments on both strands were inconsistent with the semi-discontinuous DNA replication model. Our study supported the discontinuous DNA replication model. Besides, we also observed that Okazaki fragments were overpresented in the transcribed regions in S. cerevisiae mitochondrial genome, which indicated the interplay between transcription and DNA replication. Examination of the distribution of Okazaki fragments in Saccharomyces cerevisiae strain S288C.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Wenchao Yang

PROVIDER: E-GEOD-42929 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Next-generation sequencing of Okazaki fragments extracted from Saccharomyces cerevisiae

Project description:We investigated the genome-wide distribution of Okazaki fragments in the commonly used laboratory Saccharomyces cerevisiae strain S288C to study the DNA replication model adopted by the budding yeast. The method based upon lambda exonuclease digestion for purification of RNA-primed replication intermediates was first improved to be suitable for the purification of Okazaki fragments. Then, we used this improved method to purify Okazaki fragments from S288C yeast cells, followed by Illumina sequencing. We found that the expected asymmetric distribution of Okazaki fragments around confirmed replication origins, which was derived from the semi-discontinuous DNA replication model, was not observed on S. cerevisiae chromosomes. Even around two highly efficient replication origins, ARS522 and ARS416, the ratios of Okazaki fragments on both strands were inconsistent with the semi-discontinuous DNA replication model. Our study supported the discontinuous DNA replication model. Besides, we also observed that Okazaki fragments were overpresented in the transcribed regions in S. cerevisiae mitochondrial genome, which indicated the interplay between transcription and DNA replication.

2013-06-18 | GSE42929 | GEO

Intrinsic Coupling of Lagging Strand Synthesis to Chromatin Assembly

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

Intrinsic Coupling of Lagging Strand Synthesis to Chromatin Assembly

2012-02-21 | GSE33786 | GEO

Separable recruitment of DNA Polymerase α for leading- and lagging-strand replication initiation

Project description:Okazaki fragments were purified and sequenced from asynchronous S. cerevisiae at various DNA polymerase alpha levels in the presence or absence of interactions with Ctf4. The sequencing data was used to quantify efficiencies of replication origins.

2018-06-16 | GSE115897 | GEO

Unchanged PCNA and DNMT1 dynamics during replication in DNA ligase I-deficient cells but abnormal chromatin levels of non-replicative histone H1

Project description:DNA ligase I (LigI), the predominant enzyme that joins Okazaki fragments, interacts with PCNA and Pol d. LigI also interacts with UHRF1, linking Okazaki fragment joining with DNA maintenance methylation. Okazaki fragments can also be joined by a relatively poorly characterized DNA ligase IIIa (LigIIIa)-dependent backup pathway. Here we examined the effect of LigI-deficiency on proteins at the replication fork. Notably, LigI-deficiency did not alter the kinetics of association of the PCNA clamp, the leading strand polymerase Pol e, DNA maintenance methylation proteins and core histones with newly synthesized DNA. While the absence of major changes in replication and methylation proteins is consistent with the similar proliferation rate and DNA methylation levels of the LIG1 null cells compared with the parental cells, the increased levels of LigIIIa/XRCC1 and Pol d at the replication fork and in bulk chromatin indicate that there are subtle replication defects in the absence of LigI. Interestingly, the non-replicative histone H1 variant, H1.0, is present in the chromatin of LigI-deficient mouse and human cells. This alteration was not corrected by expression of wild type LigI, suggesting that it is a stable epigenetic change that may contribute to the immunodeficiencies linked with inherited LigI-deficiency syndrome.

2023-04-04 | PXD039547 | Pride

Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo

Project description:Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5’ flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2, and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been clearly defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in S. cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5’ flaps, as predicted by some models. Further, using cyclin-controlled constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.

2018-08-04 | GSE118078 | GEO

Reduced cellular levels of DNA polymerase delta alter replication-fork dynamics and enzymology, and globally impair lagging-strand processing

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

Genome-wide replication fork directionality measured by strand-specific sequencing of Okazaki fragments in chicken lymphoma cells (DT40).

Project description:Genome-wide replication fork directionality measured by strand-specific sequencing of Okazaki fragments in chicken lymphoma cells (DT40).

2022-02-18 | GSE196761 | GEO

Replication-coupled nucleosome assembly and positioning by ATP-dependent chromatin remodeling enzymes

Project description:We have previously shown that Okazaki fragments in Saccharomyces cerevisiae are sized according to the chromatin repeat. Here we demonstrate that nucleosome positioning is rapidly established on newly synthesized DNA. Using deep sequencing, we demonstrate that ATP-dependent chromatin remodeling enzymes, Isw1 and Chd1, collaborate with histone chaperones, such as CAF-1 and Rtt106, to remodel nucleosomes, as they are loaded. Importantly, we find that nucleosome positioning is ultimately specified by select sequence-specific DNA-binding factors, which serve as physical cues for chromatin remodeling. Our results provide a mechanistic understanding of how chromatin structures are replicated in vivo, and show that chromatin structure at gene promoters is rapidly established after DNA replication. Altogether, our data provide evidence for coordinated âloading and remodelingâ of nucleosomes behind the replication fork, in collaboration with packing of nucleosomes against a barrier. 7 samples, including a wild type, are included. All are single-end sequenced via Ion Torrent PGM methodology.

2016-04-14 | E-GEOD-63583 | biostudies-arrayexpress

Replication maps of HCT116 cells grown under various conditions

Project description:Okazaki fragments were purified and sequenced from HCT116 cells. The sequencing data were used to infer sites and efficiencies of replication initiation and termination.

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