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

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE173065 | GEO | 2021/04/22

REPOSITORIES: GEO

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Project description:Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites. Chromatin immunoprecipitation of micrococcal nuclease-digested native chromatin followed by paired-end sequencing (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin 'ORGANIC' profiling) of DNA-binding proteins Abf1 and Reb1 from S. cerevisiae and GAGA-binding factor (GAF) and Pipsqueak (Psq) from D. melanogaster S2 cells; and, Sono-seq (paired-end sequencing of formaldehyde cross-linked and sonicated chromatin) of yeast nuclei. Reb1 ORGANIC profiling was performed at three different salt (NaCl) concentrations (80, 150, and 600 mM) and Abf1 ORGANIC profiling was done at two different salt concentrations (80 and 600 mM) to achieve varying levels of stringency. GAF and Psq ORGANIC profiles were determined at 80 mM salt. Two replicates each of Reb1 and Abf1 600 mM ORGANIC experiments, mixed Drosophila S2 cell and S. cerevisiae nuclei Reb1 ORGANIC experiments, yeast Sono-seq, and GAF and Psq ORGANIC experiments were performed. Each S. cerevisiae and mixed S2 cell/yeast ORGANIC profiling experiment included separately sequenced input chromatin and ChIP samples. Total of 24 samples.

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

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