Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae

Single-stranded DNA breaks (nicks) were tagged by biotin-dCTP in wild-type budding yeast cells and mapped by nick-ChIP coupled to whole-genomic microarrays. Live budding yeast cells were fixed by 1% formaldehyde and encapsulated into low-melting point agarose plugs. After cell lysis, free 3'-OH DNA termini were tagged by limited in situ nick translation using E Coli DNA pol I holoenzyme, incorporating bioin-dCTP. As a positive control of nick labeling, part of the samples were treated by nickase enzyme introducing site-specific exogenous ss breaks (nickase-treated samples). Nucleic acid was from control and nickase-treated samples and purified and fragmented by sonication (to an average size of 300-500 bp) and immunoprecipitated by anti-biotin monoclonal antibodies (Sigma). After WGA (whole genome amplification) input samples were lablelled by Cy3, IP sample were labelled by Cy5 and were co-hybridized to 4x44k Agilent microarrays.