Dataset Information

Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation.

ABSTRACT: Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome organization in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin organization defects of an ELG1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role.

SUBMITTER: Gali VK

PROVIDER: S-EPMC6258251 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation.

Gali Vamsi K VK Dickerson David D Katou Yuki Y Fujiki Katsunori K Shirahige Katsuhiko K Owen-Hughes Tom T Kubota Takashi T Donaldson Anne D AD

PLoS genetics 20181112 11

Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome organization in the absence of Elg1. Using a proteomic approach to ...[more]

PMID: 30418970

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Project description:Affinity purification coupled to mass spectrometry (AP-MS) is an effective means of identifying protein-protein interactions to better understand biological functions. However, issues associated with sample preparation still limit the success of AP-MS for specific classes of proteins, including those associated with chromatin that exhibit overall poor solubility in the protocols normally used for AP-MS analysis. Here, we wanted to provide a generally applicable method to simultaneously identify interactors for the chromatin-bound and the soluble fractions of a given bait protein. Using four FLAG-tagged canonical histone proteins (H2A, H2B, H3.1 and H4) we demonstrate that the chromatin solubility issue can be robustly alleviated by fragmenting DNA prior to AP-MS using a combination of sonication and nuclease treatment. We show that - in comparison to a commonly used AP-MS method - our optimized protocol greatly improves the recovery of chromatin-associated interactors for core histones. Critically, this is achieved while preserving the interaction partners associated with the soluble portion of the histones. Detailed protocols amenable to the study of both histone and non-histone baits are presented here.This manuscript describes workflow improvements to enable the recovery of chromatin-bound interactors by affinity purification coupled to mass spectrometry (AP-MS). This is significant, as most of the high-throughput studies to date can only monitor protein-protein interactions for soluble (not bound to chromatin) components. By consequence, we still poorly understand how protein complexes form on chromatin, which greatly hampers our understanding of gene expression. Using core histones as test cases, we show here a simple and universally applicable workflow that permits the identification of chromatin-bound protein-protein interactions. As exemplified in our manuscript, this revised protocol should result in a much deeper understanding of chromatin biology. This article is part of a Special Issue: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

Dataset Information

Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation.

Publications

Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation.

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