Preferential repair of DNA double-strand break at the active gene in vivo.
Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3' end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells.
Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine, Carbondale, Illinois 62901, USA.
Priyasri
P
Chaurasia
Rwik
R
Sen
Tej K
TK
Pandita
Sukesh R
SR
Bhaumik
D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D056426:Genetic Loci / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C516801:ADH1 protein, S cerevisiae; D000426:Alcohol Dehydrogenase; C530845:HO protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D000135:Acid Phosphatase; C488916:PHO5 protein, S cerevisiae