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Exonuclease-1 interactome and phosphorylation sites

ABSTRACT: To help shedding light on regulatory aspects of DNA repair and remodeling pathways in which EXO1 participates and to help revealing novel cellular processes in which EXO1 is involved, we set out to identify proteins interacting with EXO1. Both a small-scale approach and a comprehensive analysis of the EXO1 interactome allowed identifying novel protein partners that are involved in RNA processing or that are the causative agents of rare X-linked disorders. Follow-up biochemical studies allowed validating the direct interaction with selected proteins. Extensive coverage of EXO1 sequence (>85%) allowed conducting an in-depth analysis of its phosphorylation sites, with the identification of 26 residues that are differentially modified in untreated conditions or upon induction of DNA damage.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Daniel Hess

LAB HEAD: Daniel Hess

PROVIDER: PXD002780 | Pride | 2019-01-31

REPOSITORIES: Pride

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The human Exonuclease-1 interactome and phosphorylation sites.

Eid Wassim W Hess Daniel D König Christiane C Gentili Christian C Ferrari Stefano S

Biochemical and biophysical research communications 20190502 3

Error-free repair of DNA double-strand breaks is orchestrated by homologous recombination (HR) pathways and requires the concerted action of several factors. Among these, Exonulcease-1 (EXO1) and DNA2/BLM execute extensive resection of DNA ends to produce 3'-overhangs, which are key intermediates for downstream steps of HR. To help shedding light on regulatory aspects of DNA repair pathways in which EXO1 participates, we set out to identify proteins interacting with EXO1. Affinity purification o ...[more]

PMID: 31056253

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Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice

Project description:DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease. In mammals, it is involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1-/- and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1-/- mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1-/- mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1-/- mice was comparably defective, switch-switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1-/- mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1-/- mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.

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Meiotic double-strand break end resection mapping by S1Seq

Project description:The DNA double-strand breaks that initiate homologous recombination during meiosis are subject to extensive 5Ê¹â??3â?² exonucleolytic processing. This resection is a central and conserved feature of recombination, yet its mechanism is poorly understood. Using a purpose-made deep-sequencing method, namely S1Seq, we mapped meiotic resection endpoints genome-wide at high spatial resolution in Saccharomyces cerevisiae. Generating full-length resection tracts requires Exo1 exonuclease activity and the DNA-damage responsive kinase Tel1, but not the helicase Sgs1. Tel1 is also required for efficient and timely initiation of resection. We find that distributions of resection endpoints at individual genomic loci display pronounced heterogeneity that reflects a tendency for nucleosomes to block Exo1 in vivo, yet modeling experiments indicate that Exo1 digests chromatin with high apparent processivity and at rates approaching those for naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a determining feature of the meiotic resection landscape. 36 samples total: two biological replicate S1Seq maps of S.cerevisiae for each genetic background and time point in meiosis

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Chromatin remodeler Fun30 promotes efficient DSB end resection and recombination during yeast meiosis

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Chromatin-immunoprecipitant (ChIP) for CrdR binding site analysis in whole-genome tilling array

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2020-01-15 | GSE133108 | GEO

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Rpd3L Contributes to the DNA Damage Sensitivity of Saccharomyces cerevisiae Checkpoint Mutants

Project description:DNA replication forks that are stalled by DNA damage activate an S phase checkpoint that prevents irreversible fork arrest and cell death. The increased cell death caused by DNA damage in budding yeast cells lacking the Rad53 checkpoint protein kinase is partially suppressed by deletion of the EXO1 gene. Here,we identified that loss of the histone deacetylase complex Rpd3L promotes survival of rad53∆ cells exposed to DNA damaging agents. From epistasis analysis, we show that this suppression operates in a separate pathway from the previously described suppression by deletion of EXO1.

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Meiotic double-strand break end resection mapping by S1Seq

Project description:The DNA double-strand breaks that initiate homologous recombination during meiosis are subject to extensive 5ʹ→3′ exonucleolytic processing. This resection is a central and conserved feature of recombination, yet its mechanism is poorly understood. Using a purpose-made deep-sequencing method, namely S1Seq, we mapped meiotic resection endpoints genome-wide at high spatial resolution in Saccharomyces cerevisiae. Generating full-length resection tracts requires Exo1 exonuclease activity and the DNA-damage responsive kinase Tel1, but not the helicase Sgs1. Tel1 is also required for efficient and timely initiation of resection. We find that distributions of resection endpoints at individual genomic loci display pronounced heterogeneity that reflects a tendency for nucleosomes to block Exo1 in vivo, yet modeling experiments indicate that Exo1 digests chromatin with high apparent processivity and at rates approaching those for naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a determining feature of the meiotic resection landscape.

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