Abstract
Rad26, the yeast homologue of human Cockayne syndrome group B protein, and Rpb9, a nonessential subunit of RNA polymerase II, have been shown to mediate two subpathways of transcription-coupled DNA repair in yeast. Here we show that Rad26- and Rpb9-mediated repair in the yeast GAL1 gene is differently modulated by different promoter elements. The initiation site and efficiency of Rad26-mediated repair in the transcribed strand are determined by the upstream activating sequence (UAS) but not by the TATA or local sequences. The role of UAS in determining the Rad26-mediated repair is not through loading of RNA polymerase II or the transcriptional regulatory complex SAGA. However, both the UAS and the TATA sequences are essential for confining Rad26-mediated repair to the transcribed strand. Mutation of the TATA sequence, which greatly reduces transcription, or deletion of the TATA or mutation of the UAS, which completely abolishes transcription, causes Rad26-mediated repair to occur in both strands. Rpb9-mediated repair only occurs in the transcribed strand and is efficient only in the presence of both TATA and UAS sequences. Also, the efficiency of Rpb9-mediated repair is dependent on the SAGA complex. Our results suggest that Rad26-mediated repair can be either transcription-coupled, provided that a substantial level of transcription is present, or transcription-independent, if the transcription is too low or absent. In contrast, Rpb9-mediated repair is strictly transcription-coupled and is efficient only when the transcription level is high.
Highlights
Nucleotide excision repair (NER)2 is a conserved DNA repair mechanism that removes a wide range of bulky DNA lesions including UV light-induced cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts
Modulation of Rad26- and Rpb9-mediated Repair in the transcribed strand (TS) of To analyze Rad26- and Rpb9-mediated repairs in the GAL1 Gene by TATA and upstream activating sequence (UAS) Elements—The GAL1 gene the plasmid-borne GAL1 fragments, the 2-kb plasmid-borne is essential for yeast cells to grow in galactose media because it GAL1–10 fragments (Fig. 1A) were gel-purified following encodes a galactokinase that phosphorylates galactose to galac- digestion of the total DNA with HindIII and incised at CPDs tose-1-phosphate in the first step of galactose catabolism (29, with an excess amount of T4 endonuclease V [32]
In rad16 rad26 cells, where only the Rpb9-mediated repair is active [14, 15], mutation of the GAL1 TATA essentially abolished repair in the TS of the GAL1 gene (Fig. 2, compare C and G). These results indicate that the initiation site and efficiency of Rad26-mediated repair in the TS of the GAL1 gene are largely independent of the TATA element and transcription level
Summary
Yeast Strains and Plasmids—The wild-type yeast strain Y452 (MAT␣, ura, his, leu, leu112, cir°) and its isogenic rad, rad rpb, and rad rad deletion mutants were created as described previously [15]. A 2-kb normal GAL1–10 fragment, encompassing the UAS and 5Ј portions (0.7 kb) of each of the genes, was PCR-amplified using primers 1 and 2. Primer pairs 1 and 4 and 2 and 3 were used to amplify two fragments, which were digested with StyI and ligated to create a GAL1–10 fragment with the GAL1 TATA mutated from ATATAAA [21] to CCATGGA. Primer pairs 1 and 6 and 2 and 5 were used to amplify another two fragments, which were digested with SphI and ligated to create a GAL1–10 fragment with the UAS mutated (Table 2). Primer 1 was paired with primers 7, 8, 9, and 10 to amplify the GAL1–10 fragments with deletion from the GAL1 gene down to ϩ14, Ϫ72, Ϫ111, and Ϫ185, respectively. Ϳ1 g of total DNA was Mutations in the UAS of plasmid borne GAL1 gene Gal cognitive triplet nucleotide sequences are underlined; nucleotides mutated are shown in bold
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
