Transcription, nucleosome stability, and DNA repair in a yeast minichromosome

The template strand of the URA3 gene in the minichromosome YRpTRURAP is repaired of UV-induced cyclobutyl pyrimidine dimers (PD) much more efficiently than the nontemplate strand in growth-arrested Saccharomyces cerevisiae cells (Smerdon, M. J., and Thoma, F. (1990) Cell 61, 675). However, other regions of the plasmid are also efficiently repaired. We have examined the transcription and chromatin structure of these regions in growth-arrested cells to allow a more detailed comparison of transcription, nucleosome stability, and excision repair efficiency. Northern analysis, using strand-specific probes, indicates that four different transcripts are made from YRpTRURAP in addition to the URA3 mRNA in both growing and growth-arrested cells. The templates for these transcripts encompass all of the efficiently repaired regions outside of the URA3 gene. Nucleosome mapping indicates that the structure of the minichromosome in growth-arrested cells is indistinguishable from that of growing cells except for two nucleosomes in the region 500-800 base pairs 5' of the URA3 gene which become much less stable in growth-arrested cells. Comparison of the distribution of YRpTRURAP topoisomers in the two states, however, indicates that these nucleosomes are not lost from the majority of plasmid molecules. One of the four transcripts initiates in this region and increases by more than 5-fold in growth-arrested cells. Another transcript extends into a "slowly repaired" region which contains a very stable nucleosome. By determining the stability and relative amounts (at equilibrium) of the RNAs made from YRpTRURAP, transcription rates were determined and compared with the average PD repair rate for the different template regions. The results indicate that: 1) the rate of excision repair increases once a low, basal rate of transcription is achieved; 2) beyond this rate of transcription there is no simple correlation between the rates of transcription and PD repair; 3) nucleosome stability may "override" the coupling between transcription and repair if the transcription rate is low; and 4) at higher transcription rates, repair may be insensitive to nucleosome stability.