Dataset Information

Identification of a new XPF-ERCC1-like complex important for meiotic crossovers production

ABSTRACT: Proteins of the XPF-ERCC1 complex family play roles in DNA repair. Known members (four in mammals, two in budding yeast) recognize branched DNA structures and most bear nuclease activity. Here, we identified a new XPF-ERCC1-like complex important for meiotic crossovers production. Through a proteomic screen, we found that Zip2 and Spo16, two proteins important for CO formation in budding yeast, form a complex and share structural similarities with XPF-ERCC1. Zip2 XPF domain is important for CO formation and, in complex with Spo16, preferentially binds branched DNA molecules. However, Zip2-Spo16 lacks endonucleolytic activity. This suggests that Zip2-Spo16 works as a structural module that recognizes and stabilizes joint molecules to ensure CO formation. Moreover, Zip2-Spo16 forms a complex (called ZZS) with Zip4, another protein important for CO formation, which directly interacts with components of the chromosome axis, providing a link between recombination intermediates and the underlying chromosome structure.

OTHER RELATED OMICS DATASETS IN: PRJNA407372

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Yohann Couté

LAB HEAD: Virginie Brun

PROVIDER: PXD007735 | Pride | 2018-04-10

REPOSITORIES: Pride

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Publications

A meiotic XPF-ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation.

De Muyt Arnaud A Pyatnitskaya Alexandra A Andréani Jessica J Ranjha Lepakshi L Ramus Claire C Laureau Raphaëlle R Fernandez-Vega Ambra A Holoch Daniel D Girard Elodie E Govin Jérome J Margueron Raphaël R Couté Yohann Y Cejka Petr P Guérois Raphaël R Borde Valérie V

Genes & development 20180209 3-4

Meiotic crossover formation requires the stabilization of early recombination intermediates by a set of proteins and occurs within the environment of the chromosome axis, a structure important for the regulation of meiotic recombination events. The molecular mechanisms underlying and connecting crossover recombination and axis localization are elusive. Here, we identified the ZZS (Zip2-Zip4-Spo16) complex, required for crossover formation, which carries two distinct activities: one provided by Z ...[more]

PMID: 29440262

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Project description:We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage. The RNET-seq method involves trimming of excess nascent RNA from the 5’ ends leaving only the nascent RNA that is protected by RNAP. A previous in vitro study showed that an RNAP forming an EC protects different lengths of the 3’-proximal transcript from trimming by RNases A and T1 depending on the EC translocation state. Post-translocated, pre-translocated, and backtracked complexes protect 14-nt, 15-nt and >15-nt segments of the RNA, respectively. Because the very 3’ end of the RNA is extruded to a narrow pore within front of the enzyme during backtracking, the extruded RNA remains inaccessible to RNases increasing in length as backtracking increases. Thus, paused RNAP in either the pre- and post-translocated states as well as in different backtracked distances were monitored over the entire genome. The unique properties of our RNET-seq approach provided an opportunity to dissect the core mechanisms of different types of pausing in living cells.

Dataset Information

Identification of a new XPF-ERCC1-like complex important for meiotic crossovers production

Publications

A meiotic XPF-ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation.

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