Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ACKNOWLEDGMENTS We are grateful to J ¨urg Kohli, Ramsay J. McFarlane, Paul Russell, Gerald R. Smith, Walter W. Steiner and the National BioResource Project (NBRP) Japan for providing strains and to C. Bryer for technical assistance. FUNDING Wellcome Trust [090767/Z/09/Z to M.C.W.]; College of Life Sciences and Medicine, University of Aberdeen [to A.L., in part]. Funding for open access charge: Wellcome TrustPeer reviewedPublisher PD

Phosphoregulation of DNA repair via the Rad51 auxiliary factor Swi5–Sfr1

Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks, the most severe form of DNA damage. The Rad51 protein is central to HR, but multiple auxiliary factors regulate its activity. The heterodimeric Swi5–Sfr1 complex is one such factor. It was previously shown that two sites within the intrinsically disordered domain of Sfr1 are critical for the interaction with Rad51. Here, we show that phosphorylation of five residues within this domain regulates the interaction of Swi5–Sfr1 with Rad51. Biochemical reconstitutions demonstrated that a phosphomimetic mutant version of Swi5–Sfr1 is defective in both the physical and functional interaction with Rad51. This translated to a defect in DNA repair, with the phosphomimetic mutant yeast strain phenocopying a previously established interaction mutant. Interestingly, a strain in which Sfr1 phosphorylation was blocked also displayed sensitivity to DNA damage. Taken together, we propose that controlled phosphorylation of Sfr1 is important for the role of Swi5–Sfr1 in promoting Rad51-dependent DNA repair

Homologous Recombination under the Single-Molecule Fluorescence Microscope

Homologous recombination (HR) is a complex biological process and is central to meiosis and for repair of DNA double-strand breaks. Although the HR process has been the subject of intensive study for more than three decades, the complex protein–protein and protein–DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA which is difficult to capture by routine biochemical or structural biology methods. In recent years, single-molecule fluorescence microscopy has been a popular method in the field of HR to visualize these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. In this review, we describe recent efforts that employ single-molecule fluorescence microscopy to investigate protein–protein and protein–DNA interactions operating on three key DNA-substrates: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and four-way DNA called Holliday junction (HJ). We also outline the technological advances and several key insights revealed by these studies in terms of protein assembly on these DNA substrates and highlight the foreseeable promise of single-molecule fluorescence microscopy in advancing our understanding of homologous recombination

Phosphoregulation of DNA repair via the Rad51 auxiliary factor Swi5-Sfr1

Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks, the most severe form of DNA damage. The Rad51 protein is central to HR, but multiple auxiliary factors regulate its activity. The heterodimeric Swi5-Sfr1 complex is one such factor. It was previously shown that two sites within the intrinsically disordered domain of Sfr1 are critical for the interaction with Rad51. Here, we show that phosphorylation of five residues within this domain regulates the interaction of Swi5-Sfr1 with Rad51. Biochemical reconstitutions demonstrated that a phosphomimetic mutant version of Swi5-Sfr1 is defective in both the physical and functional interaction with Rad51. This translated to a defect in DNA repair, with the phosphomimetic mutant yeast strain phenocopying the previously established interaction mutant. Interestingly, a strain in which Sfr1 phosphorylation was blocked also displayed sensitivity to DNA damage. Taken together, we propose that controlled phosphorylation of Sfr1 is important for the role of Swi5-Sfr1 in promoting Rad51-dependent DNA repair

Analyses biochimiques de la recombinaison homologue méiotique chez schizosaccharomyces pombe

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2009-2010Les cassures double-brin de l'acide désoxyribonucléique (ADN) sont parmi les lésions les plus cytotoxiques car une seule lésion non réparée est létale chez la levure. Dans le but de réparer efficacement ces dommages, la cellule dispose de différents mécanismes tels que le Non Homologous End Joining (NHEJ). Toutefois ces mécanismes peuvent causer des mutations et, lorsqu' il est possible, la cellule privilégie un système qui permet une réparation très fiable: la recombinaison homologue. Ce processus peut aussi être utilisé lors de la méiose pour créer de la diversité génétique. La méiose est un mécanisme complexe et une mauvaise régulation peut mener à des problèmes tels que l' aneuploïdie. La recombinaison homologue méiotique se divise en quatre étapes majeures: (i) l' initiation qui crée des cassures double-brin par un complexe muItiprotéique incluant Rec12; (ii) la résection de l'ADN ou les protéines majeures sont Rad32/Rad50/Nbsl; (iii) l'invasion d'un duplex homologue avec les protéines RadSl et Dmcl, et pour terminer (iv) la résolution des jonctions de Holliday. Lors de mes études de doctorat, je me suis concentré sur la caractérisation biochimique des principales protéines des trois premières étapes (initiation, résection et particulièrement l'invasion) de la recombinaison méiotique chez Schizosaccharomyces pombe permettant la réparation des cassures double-brin. Mes travaux avaient pour but de caractériser les protéines Dmcl et le complexe Hop2/Mndl chez S.pombe. Nous avons déterminé que Dmcl avait comme Rad 51 la capacité de former un filament hélical sur l'ADN simple brin, ansi que de catalyser des réactions d'échange de brin. D'autre part j'ai mis en évidence le rôle que joue le complexe Hop2/Mndl dans la stimulation de Dmc 1, ainsi que les différences entre les protéines de S.pombe et de la SOurIS

Mechanics and execution of homologous recombination: a single-molecule view

Homologous recombination (HR) is an essential mechanism for the repair of toxic DNA double-strand breaks (DSBs), which, when not repaired accurately, can give rise to cancer and hereditary disorders. During HR, RAD51 forms helical nucleoprotein filaments on RPA-coated ssDNA with the help of mediator proteins (BRCA2 and RAD51 paralogs) and catalyses strand invasion into homologous duplex DNA. How this is achieved in not completely understood. To dissect the process on molecular level, I first reconstituted nematode RAD-51 presynaptic filament assembly in the presence of mediator proteins at the single-molecule level and demonstrated that BRC-2 promotes RAD-51 nucleation, while RAD-51 paralogs transiently bind 5’ RAD-51 filament ends to stimulate RAD-51 growth in a 3’ to 5’ direction. In the second part of the thesis, I investigated the consequences of a permanently ‘switching on’ RAD-51 by engineering a variant of human RAD51, I287T, that forms presynaptic complexes efficiently without the recombination mediators present and analysed its impact on cellular DNA metabolism. I showed that RAD51 I287T is toxic in cells as it interferes with genome duplication by promiscuously loading at replication forks. Lastly, I demonstrated that nematode RAD-51 is surprisingly tolerant to mismatches during DNA strand exchange catalysis. The mismatch tolerance can be abolished by engineering specific mutations into the DNA binding loop of RAD-51, which causes meiotic HR stalling in the absence of regulatory motor proteins. Together, this work has uncovered unappreciated mechanisms that promote and maintain optimal RAD51 filament assembly and how deviations to optimal assembly rates can lead to disease - a phenomenon referred to as the ‘Goldilocks principle’ of RAD51 assembly.Open Acces

Main steps in DNA double-strand break repair: An introduction to homologous recombination and related processes

DNA double-strand breaks arise accidentally upon exposure of DNA to radiation, chemicals or result from faulty DNA metabolic processes. DNA breaks can also be introduced in a programmed manner, such as during the maturation of the immune system, meiosis or cancer chemo- or radiotherapy. Cells have developed a variety of repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly, vegetative cells employ mechanisms that restore the integrity of broken DNA with the highest efficiency at the lowest cost of mutagenesis. In contrast, meiotic cells or developing lymphocytes exploit DNA breakage to generate diversity. Here, we review the main pathways of eukaryotic DNA double-strand break repair with the focus on homologous recombination and its various sub-pathways. We highlight the differences between homologous recombination and end-joining mechanisms including nonhomologous end-joining and microhomology-mediated end-joining, and offer insights into how these pathways are regulated. Finally, we introduce non-canonical functions of the recombination proteins, in particular during DNA replication stress