Role of the Srs2-Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Srs2, in addition to its well-documented antirecombination activity, has been proposed to play a role in promoting synthesis-dependent strand annealing (SDSA). Here we report the identification and characterization of an SRS2 mutant with a single amino acid substitution (srs2-F891A) that specifically affects the Srs2 pro-SDSA function. This residue is located within the Srs2-Rad51 interaction domain and embedded within a protein sequence resembling a BRC repeat motif. The srs2-F891A mutation leads to a complete loss of interaction with Rad51 as measured through yeast two-hybrid analysis and a partial loss of interaction as determined through protein pull-down assays with purified Srs2, Srs2-F891A, and Rad51 proteins. Even though previous work has shown that internal deletions of the Srs2-Rad51 interaction domain block Srs2 antirecombination activity in vitro, the Srs2-F891A mutant protein, despite its weakened interaction with Rad51, exhibits no measurable defect in antirecombination activity in vitro or in vivo Surprisingly, srs2-F891A shows a robust shift from noncrossover to crossover repair products in a plasmid-based gap repair assay, but not in an ectopic physical recombination assay. Our findings suggest that the Srs2 C-terminal Rad51 interaction domain is more complex than previously thought, containing multiple interaction sites with unique effects on Srs2 activity

Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops.

Synthesis-dependent strand annealing (SDSA) is the preferred mode of homologous recombination in somatic cells leading to an obligatory non-crossover outcome, thus avoiding the potential for chromosomal rearrangements and loss of heterozygosity. Genetic analysis identified the Srs2 helicase as a prime candidate to promote SDSA. Here, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion and with a distinct polarity. Specifically, we partly reconstitute the SDSA pathway using Rad51, Rad54, RPA, RFC, DNA Polymerase δ with different forms of PCNA. Consistent with genetic data showing the requirement for SUMO and PCNA binding for the SDSA role of Srs2, Srs2 displays a slight but significant preference to disrupt extending D-loops over unextended D-loops when SUMOylated PCNA is present, compared to unmodified PCNA or monoubiquitinated PCNA. Our data establish a biochemical mechanism for the role of Srs2 in crossover suppression by promoting SDSA through disruption of extended D-loops

Etude du rôle de l'hélicase Srs2 dans la régulation de la recombinaison homologue chez la levure Saccharomyces cerevisiae

La recombinaison homologue (RH) est nécessaire au maintien de l'intégrité du génome. Cependant, elle peut parfois se révéler dangereuse en formant des structures toxiques ou en générant des réarrangements chromosomiques néfastes. Chez S. cerevisiae, l hélicase Srs2 est capable d inhiber la RH via l élimination de la protéine Rad51 de l ADNsb.Deux nouveaux mutants srs2R1 et srs2R3 ont été identifiés au laboratoire. Les résultats génétiques et biochimiques obtenus suggèrent qu ils ne sont plus capables d inhiber la recombinaison homologue à la fourche de réplication. Les données montrent de plus que le recrutement de Srs2 par PCNA sumoylé n est pas essentiel à l élimination des intermédiaires toxiques de recombinaison. Srs2 est également nécessaire pour la régulation des étapes tardives de la RH. En effet, Srs2 inhibe fortement la formation de crossing over probablement en favorisant une voie de résolution appelée SDSA. Nous montrons dans cette étude que Srs2 purifié possède in vitro l ensemble des activités biochimiques requises pour permettre l utilisation du SDSA.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Suppression of Homologous and Homeologous Recombination by the Bacterial MutS2 Protein

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The Srs2 helicase activity is stimulated by Rad51 Filaments on dsDNA : Implications for crossover incidence during mitotic recombination.

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The forkhead DNA-binding domain binds specific G2-rich RNA sequences

International audienceAbstract Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed of dozens of paralogs in mammals. The forkhead domain (FHD) is a segment of about 100 amino acids that binds an A-rich DNA sequence. Using DNA and RNA PCR-SELEX, we show that recombinant FOXL2 proteins, either wild-type or carrying the oncogenic variant C134W, recognize similar DNA-binding sites. This suggests that the oncogenic variant does not alter the intrinsic sequence-specificity of FOXL2. Most importantly, we show that FOXL2 binds G2-rich RNA sequences whereas it virtually fails to bind similar sequences in DNA chemistry. Interestingly, a statistically significant subset of genes responding to the knock-down of FOXL2/Foxl2 harbor such G2-rich sequences and are involved in crucial signaling pathways and cellular processes. In addition, we show that FOXA1, FOXO3a and chimeric FOXL2 proteins containing the FHD of the former are also able to interact with some of the preferred FOXL2-binding sequences. Our results point to an unexpected and novel characteristic of the forkhead domain, the biological relevance of which remains to be explored

Correction: The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ.

[This corrects the article DOI: 10.1371/journal.pgen.1007119.]

Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

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Structural basis for the substrate selectivity of Helicobacter pylori NucT nuclease activity

The Phospholipase D (PLD) superfamily of proteins includes a group of enzymes with nuclease activity on various nucleic acid substrates. Here, with the aim of better understanding the substrate specificity determinants in this subfamily, we have characterised the enzymatic activity and the crystal structure of NucT, a nuclease implicated in Helicobacter pylori purine salvage and natural transformation and compared them to those of its bacterial and mammalian homologues. NucT exhibits an endonuclease activity with a strong preference for single stranded nucleic acids substrates. We identified histidine124 as essential for the catalytic activity of the protein. Comparison of the NucT crystal structure at 1.58 angstrom resolution reported here with those of other members of the sub-family suggests that the specificity of NucT for single-stranded nucleic acids is provided by the width of a positively charged groove giving access to the catalytic site