Abstract
DNA replication defects are an underlying cause of genome instability, which could stem from alterations in replication intermediates such as extensive single-stranded DNA (ssDNA). Under replication stress, ssDNA is a precursor of the ultimate double-strand breaks (DSBs). Indeed, mutations that render the cell incapable of mediating and protecting the replication forks produce ssDNA genome-wide at high frequency and cause lethality when encountering DNA damage or replication perturbation. Here we describe two related microarray-based methods to query genetically unstable yeast cultures, such as the mec1 and rad53 mutants. These mutants are defective in central protein kinases in the checkpoint pathway. To induce ssDNA and DSB formation in these mutants, we utilize hydroxyurea, a drug that causes nucleotide shortage in the cell.
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Acknowledgements
This work was supported by NIH grant 4R00GM08137804 to W.F. and NIGMS grant 18926 to M.K.R.
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© 2014 Springer Science+Business Media New York
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Peng, J., Raghuraman, M.K., Feng, W. (2014). Analysis of ssDNA Gaps and DSBs in Genetically Unstable Yeast Cultures. In: Noguchi, E., Gadaleta, M. (eds) Cell Cycle Control. Methods in Molecular Biology, vol 1170. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0888-2_27
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DOI: https://doi.org/10.1007/978-1-4939-0888-2_27
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0887-5
Online ISBN: 978-1-4939-0888-2
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