Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.European Research Council ERC2014-ADG669898 TARLOOPMinisterio de Economía y Competitividad BFU2016-75058-PJunta de Andalucía BIO123

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies

Chromatin Dynamics In Homology Directed Repair

This thesis consist of six chapters and the appendices. Each chapter starts with a brief summary page. If parts or the whole of the chapter has been published, that sheet indicates the title, author list and the date of the publication as well as my personal contribution to the papers. Chapter I is an overview of chromatin organization and dynamics in the context of DNA double-strand break (DSB) repair. Chapter II is a specific published guideline for DNA recombination and repair studies. This chapter is an extracted section of my contribution to a published review. It describes the method I used to visualize and quantify chromosomal dynamics upon DNA double strand breaks in yeast S. cerevisiae. Chapter III describes my main thesis research question, results, discussion and experimental procedures. This study show that DNA damage induced histone depletion enhances homology search through the induced chromatin expansion and ectopic locus mobility, independently of local DSB movement. Moreover, we show that local DSB dynamics is cell cycle dependent and is regulated by Cohesin turnover. Finally, we find that centromeres do not detach upon DNA damage. Chapter IV in an overview of the roles of two post-translational modifications (PTMs) in the regulation of DNA repair pathway choice in budding yeast: Ubiquitination and the small ubiquitin-related modifier protein (SUMO). This chapter introduces the research context to Chapter V. Chapter V contains a study to which I had contributed in which we reported that the presence of telomeric repeat sequences on one side of a double-strand break alters the outcome of repair. We show that the two sides of the break show uncoordinated movement and are repaired asymmetrically leading to translocation. We observed that the repair outcome is tightly controlled by SUMO targeted ubiquitin ligases. Chapter VI summarizes the main conclusion of this thesis and discusses the results stemming from Chapter III together with relevant future directions. Finally, the appendices contain a list of abbreviations, my curriculum vitae and acknowledgments

Asymmetric Processing of DNA Ends at a Double-Strand Break Leads to Unconstrained Dynamics and Ectopic Translocation

Summary: Multiple pathways regulate the repair of double-strand breaks (DSBs) to suppress potentially dangerous ectopic recombination. Both sequence and chromatin context are thought to influence pathway choice between non-homologous end-joining (NHEJ) and homology-driven recombination. To test the effect of repetitive sequences on break processing, we have inserted TG-rich repeats on one side of an inducible DSB at the budding yeast MAT locus on chromosome III. Five clustered Rap1 sites within a break-proximal TG repeat are sufficient to block Mre11-Rad50-Xrs2 recruitment, impair resection, and favor elongation by telomerase. The two sides of the break lose end-to-end tethering and show enhanced, uncoordinated movement. Only the TG-free side is resected and shifts to the nuclear periphery. In contrast to persistent DSBs without TG repeats that are repaired by imprecise NHEJ, nearly all survivors of repeat-proximal DSBs repair the break by a homology-driven, non-reciprocal translocation from ChrIII-R to ChrVII-L. This suppression of imprecise NHEJ at TG-repeat-flanked DSBs requires the Uls1 translocase activity. : Marcomini et al. show that the presence of interstitial telomeric repeat sequences near a double-strand break alters the outcome of repair. A TG-flanked break loads MRX asymmetrically, supports resection only on one side, and allows uncoordinated movement of the break ends. The resected TG-free end invades homology on another chromosome driving a unidirectional translocation event. Keywords: double-strand break repair, interstitial repeat sequences, telomeres, end resection, homology-driven recombination, imprecise non-homologous end joining, MRX, Uls