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Detecting repair intermediates in vivo: Effects of DNA damage response genes on single-stranded DNA accumulation at uncapped telomeres in budding yeast

Lookup NU author(s): Dr Mikhajlo Zubko, Dr Laura MaringeleORCiD, Steven Foster, Professor David Lydall

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Abstract

Single-stranded DNA (ssDNA) is an important intermediate in many DNA repair pathways. Here we describe protocols that permit the measurement of ssDNA that has arisen in the yeast genome in vivo, in response to telomere uncapping. Yeast strains defective in DNA damage response (DDR) genes can be used to infer the roles of the corresponding proteins in regulating ssDNA production and in responding to ssDNA. Using column based methods to purify yeast genomic DNA and quantitative amplification of single-stranded DNA (QAOS) it is possible to measure ssDNA at numerous single copy loci in the yeast genome. We describe how to measure ssDNA in synchronous cultures of cdc13-1 mutants, containing a temperature sensitive mutation in an essential telomere capping protein, and in asynchronous cultures of yku70 Delta mutants also defective in telomere capping.

Publication metadata

Author(s): Zubko MK, Maringele L, Foster SS, Lydall D

Publication type: Article

Publication status: Published

Journal: Methods in Enzymology

Year: 2006

Volume: 409

Pages: 285-300

ISSN (print): 0076-6879

ISSN (electronic): 1557-7988

Publisher: Academic Press

URL: http://dx.doi.org/10.1016/S0076-6879(05)09016-6

DOI: 10.1016/S0076-6879(05)09016-6

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