title: Analysis of ssDNA gaps and DSBs in genetically unstable yeast cultures.
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.
authors.affiliation: Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, 4287 Weiskotten Hall, 750 East Adams Street, Syracuse, NY, 13210, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Peng
authors.forename: M K
authors.initials: MK
authors.lastname: Raghuraman
authors.forename: Wenyi
authors.initials: W
authors.lastname: Feng
mesh_terms: D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D018797:Cell Cycle Proteins; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000032:analysis / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D015003:Yeasts / Q000235:genetics* / Q000254:growth & development
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004277:DNA, Single-Stranded