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Intrinsic Coupling of Lagging Strand Synthesis to Chromatin Assembly

ABSTRACT: 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

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Iestyn Whitehouse

PROVIDER: E-GEOD-33786 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

2012-02-21 | GSE33786 | GEO

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

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

Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae

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

Quantitative, genome-wide analysis of eukaryotic replication initiation and termination

Project description:Via the deep-sequencing of Okazaki fragments from Saccharomyces cerevisiae, we report the first comprehensive documentation of genome-wide replication directionality in any eukaryote; this permits the systematic analysis of both replication initiation and termination. We conduct a genome-wide analysis of origin competence and efficiency, and conclude that the majority of origins are competent to fire in each cell cycle and generally do so with high efficiency. Additionally, the spatial resolution of our data allow us to determine that leading-strand initiation generally occurs within the nucleosome-free region at origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing and that the replication profile is determined largely by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture. 5 samples are included. Two are replicate, paired-end, wild-type samples sequenced via Illumina methodology. The raw data for these two are also deposited in the GEO repository under accession numbers GSM835650 and GSM835651. Two replicate, single-end, Sld2, Sld3, Dbf4, Dpb11, Cdc45 and Sld7 (SSDDCS) overexpression via galactose induction experiments are reported sequenced via Ion Torrent methodology. One single-end, Sld2, Sld3, Dbf4, Dpb11, Cdc45 and Sld7 (SSDDCS) normal expression control via glucose media experiment is reported sequenced via Ion Torrent methodology.

2013-02-28 | E-GEOD-40696 | biostudies-arrayexpress

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

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

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

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

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

Genomewide nucleotide-resolution mapping of single-strand breaks and lesions by GLOE-Seq [digestion]

Project description:Numerous methods are available for the mapping of DNA lesions ranging from double-strand breaks (DSBs) to incorporated ribonucleotides. We now present a technology based on the Genomewide Ligation of 3’-OH Ends (GLOE-Seq) and an associated computational pipeline designed for single-stranded breaks (SSBs), but versatile enough to be applied to any lesion that is convertible into a free 3’-OH terminus. We demonstrate its applicability to the mapping of Okazaki fragments without prior size selection and detect biases and asymmetries in the distribution of spontaneous SSBs in budding yeast and human DNA.

2020-04-21 | GSE134219 | GEO

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