Generation of 3′ single-stranded DNA (ssDNA) tails at the ends of a double-strand break (DSB) is essential to repair the break through accurate homology-mediated repair pathways. Several methods have been developed to measure ssDNA accumulation at a DSB in the budding yeast Saccharomyces cerevisiae. Here, we describe one of these assays, which is based on the inability of restriction enzymes to cleave ssDNA. Digestion of genomic DNA prepared at different time points after DSB generation leads to the formation of ssDNA fragments whose length increases as the 5′ strand degradation proceeds beyond restriction sites. After the separation by electrophoresis on alkaline denaturing agarose gel, these ssDNA fragments can be visualized by hybridization with an RNA probe that anneals with the 3′-undegraded DSB strand. This assay allows a direct and comprehensive visualization of DSB end processing.

Colombo, C., Menin, L., & Clerici, M. (2018). Alkaline denaturing southern blot analysis to monitor double-strand break processing. In M. Muzi Falconi, & G.W. Brown (a cura di), Genome Instability : Methods and Protocols (pp. 131-145). Humana Press Inc. [10.1007/978-1-4939-7306-4_11].

Alkaline denaturing southern blot analysis to monitor double-strand break processing

Colombo, CV
Primo
;
Menin, L
Secondo
;
Clerici, M
Ultimo
2018

Abstract

Generation of 3′ single-stranded DNA (ssDNA) tails at the ends of a double-strand break (DSB) is essential to repair the break through accurate homology-mediated repair pathways. Several methods have been developed to measure ssDNA accumulation at a DSB in the budding yeast Saccharomyces cerevisiae. Here, we describe one of these assays, which is based on the inability of restriction enzymes to cleave ssDNA. Digestion of genomic DNA prepared at different time points after DSB generation leads to the formation of ssDNA fragments whose length increases as the 5′ strand degradation proceeds beyond restriction sites. After the separation by electrophoresis on alkaline denaturing agarose gel, these ssDNA fragments can be visualized by hybridization with an RNA probe that anneals with the 3′-undegraded DSB strand. This assay allows a direct and comprehensive visualization of DSB end processing.
No
Scientifica
Capitolo o saggio
Alkaline denaturing conditions; DNA double-strand breaks; Electrophoresis; HO endonuclease; MAT locus; Resection; RNA probe; Single-stranded DNA; Southern blot;
Alkaline denaturing conditions; DNA double-strand breaks; Electrophoresis; HO endonuclease; MAT locus; Resection; RNA probe; Single-stranded DNA; Southern blot; Molecular Biology; Genetics
English
Genome Instability : Methods and Protocols
978-1-4939-7305-7
Colombo, C., Menin, L., & Clerici, M. (2018). Alkaline denaturing southern blot analysis to monitor double-strand break processing. In M. Muzi Falconi, & G.W. Brown (a cura di), Genome Instability : Methods and Protocols (pp. 131-145). Humana Press Inc. [10.1007/978-1-4939-7306-4_11].
Colombo, C; Menin, L; Clerici, M
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/183056
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