Transitions in the coupling of transcription and nucleotide excision repair within RNA polymerase II-transcribed genes of Saccharomyces cerevisiae

Abstract

The molecular mechanism of transcription-coupled nucleotide excision repair in eukaryotes is poorly understood. The identification of the dual role of basal transcription factor TFIIH in DNA repair and transcription provided a plausible link between both processes. However, TFIIH is not part of the elongating transcription complex, suggesting that additional components are required to recruit TFIIH when RNA polymerase II (RNAPII) stalls at the site of DNA damage. Previously, we have shown that the yeast Rad26 protein is involved in transcription-coupled DNA repair. This paper describes the differential contribution of the Rad26 protein to efficient removal of UV-induced cyclobutane pyrimidine dimers (CPDs) from transcribed DNA. Two distinct regions within the transcribed strand of RNAPII-transcribed genes are identified that differ in their requirement for the RAD26 gene product. Using high-resolution repair analysis, we determined the in vivo repair kinetics of cyclobutane pyrimidine dimers positioned around the transcription initiation site of RNAPII-transcribed genes RPB2 and URA3. Although transcription-coupled repair is severely reduced in rad26 mutants, lesions positioned in a small region immediately downstream of transcription initiation are efficiently removed in the absence of Rad26. The observed transition in repair characteristics is abrupt and in excellent agreement with the region where TFIIH dissociates from RNAPII in vitro, strongly suggesting an inverse correlation between TFIIH association and Rad26 requirement. These data suggest that a transcription repair coupling factor (Rad26/CSB) is required for efficient repair only during the elongating stages of RNAPII transcription.

Figure 1

Repair of UV-induced CPDs at single nucleotide resolution along the transcription initiation site of the S. cerevisiae RPB2 locus. Data are for the template DNA strand, nucleotides −40 to +200 with respect to the start site of transcription. (a) Wild-type RAD⁺, (b) isogenic rad7Δ, and (c) rad7Δrad26Δ cells were irradiated with 70 J/m², and repair was allowed for 0, 20, and 40 min. The large arrow indicates the major transcription initiation site and the direction of transcription. Samples mock-treated or treated with the dimer-specific enzyme T4endoV are denoted − and +, respectively.

Figure 2

Graphic representation of quantified repair rates for the template strand of the URA3 locus from position −50 to +100 in (a) repair proficient RAD⁺ background and in isogenic (b) rad7Δ, (c) rad26Δ, and (d) rad7Δrad26Δ mutant strains. Repair half-time values, determined as the time at which 50% of the initial CPD signal was removed, were calculated for each individual CPD position and depicted above its corresponding dipyrimidine position. Repair t½ = U indicates that CPDs were unrepaired after 2 hr of incubation.

Figure 3

Repair of UV-induced CPDs at single nucleotide resolution along the template (a) and the nontemplate strand (b) of the S. cerevisiae URA3 locus in a rad26rad7 strain. Cells were irradiated with 70 J/m², and repair was allowed for 0, 20, 40, and 80 min. The large arrow indicates the major transcription initiation site and the direction of transcription. Samples mock-treated or treated with the dimer-specific enzyme T4endoV are denoted − and +, respectively. More DNA was present in b. t = 40.

Figure 4

Graphic representation of NER for the URA3 transcribed strand in (a) rad28, (b) rad14, and (c) rad3–2 genetic background. Data for the rad28 mutant were obtained with time samples of 0, 5, 10, 15, and 20 min of incubation after irradiation whereas 0, 20-, 40-, 60-, and 120-min time samples were used for repair analysis of the rad14 and rad3–2 mutants. Repair half-time values, determined as the time at which 50% of the initial CPD signal was removed were calculated for each individual CPD position and depicted above its corresponding dipyrimidine position. Repair t½ = U indicates that CPDs were unrepaired after 2 hr of incubation.