Nucleotide excision repair in a constitutive and inducible gene of a yeast minichromosome in intact cells.
Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in a yeast minichromosome, having a galactose-inducible GAL1:URA3 fusion gene, a constitutively expressed HIS3 gene and varied regions of chromatin structure. Transcription of GAL1:URA3 increased >150-fold, while HIS3 expression decreased <2-fold when cells were switched from glucose to galactose medium. Following galactose induction, four nucleosomes were displaced or rearranged in the GAL3-GAL10 region. However, no change in nucleosome arrangement was observed in other regions of the minichromosome following induction, indicating that only a few plasmid molecules actively transcribe at any one time. Repair at 269 cis-syn CPD sites revealed moderate preferential repair of the transcribed strand of GAL1:URA3 in galactose, consistent with transcription-coupled repair in a fraction of these genes. Many sites upstream of the transcription start site in the transcribed strand were also repaired faster upon induction. There is remarkable repair heterogeneity in the HIS3 gene and preferential repair is seen only in a short sequence immediately downstream of the transcription start site. Finally, a mild correlation of repair heterogeneity with nucleosome positions was observed in the transcribed strand of the inactive GAL1:URA3 gene and this correlation was abolished upon galactose induction.
Li
S
S
Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-4660, USA,
Livingstone-Zatchej
M
M
Gupta
R
R
Meijer
M
M
Thoma
F
F
Smerdon
M J
MJ
D002843:Chromatin / Q000378:metabolism; D015825:Chromosomes, Fungal / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D004260:DNA Repair; D004271:DNA, Fungal / Q000032:analysis / Q000528:radiation effects; D017930:Genes, Reporter / Q000235:genetics; D008957:Models, Genetic; D010957:Plasmids; D011740:Pyrimidine Dimers / Q000378:metabolism*; D012331:RNA, Fungal / Q000032:analysis / Q000528:radiation effects; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D002843:Chromatin; D004271:DNA, Fungal; D011740:Pyrimidine Dimers; D012331:RNA, Fungal