Titlebook: Homologous Recombination; Methods and Protocol Andrés Aguilera,Aura Carreira Book 2021 Springer Science+Business Media, LLC, part of Spring

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,Detection of DNA Double-Strand Breaks by γ-H2AX Immunodetection,tion of DSBs with different diseases and its potential to kill cells. DSB levels can be obtained by measuring levels of γ-H2AX in extracts of cell populations or by counting foci in individual nuclei. In this chapter some techniques to detect γ-H2AX are described.

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Quantifying DNA End Resection in Human Cells,thods. This assay, excluding the time for making the stable cell line expressing the restriction enzyme .SI fused to the estrogen receptor hormone-binding domain (ER-.SI), can be completed within 3 days.

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Intrachromosomal Recombination in Yeast,n the two defective copies can result in a wild-type gene and a prototrophic phenotype for the cell. Methods to use these types of reporters to determine recombination rates between the two gene copies are described, and their use in monitoring both increased and decreased recombinations is discussed.

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Branch Migration Activity of Rad54 Protein,ctions with high specificity and promotes their branch migration in an ATP hydrolysis-dependent manner. Here we describe the methods our laboratory used to characterize the branch migration activity of Rad54. These assays are applicable for other branch migration proteins regardless of whether they have canonical helicase activity or not.

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Monitoring Gene Conversion in Budding Yeast by Southern Blot Analysis,logous recombination. A method is described using an ectopic homologous donor sequence to repair an HO endonuclease-induced DSB. These gene conversion events can occur with or without crossing-over, the products of which are distinguished as different-sized restriction endonuclease fragments. The method of Southern blotting is described in detail.

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