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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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE33786 | GEO | 2012/02/21

SECONDARY ACCESSION(S): PRJNA148051

REPOSITORIES: GEO

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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. 4 samples: replicate samples of wild-type and pol32 knockout

2012-02-21 | E-GEOD-33786 | biostudies-arrayexpress

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

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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.

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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.

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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.

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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.

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2023-04-04 | PXD039547 | Pride

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