RNA polymerase II transcription suppresses nucleosomal modulation of UV-induced (6-4) photoproduct and cyclobutane pyrimidine dimer repair in yeast

Abstract

The nucleotide excision repair (NER) pathway is able to remove a wide variety of structurally unrelated lesions from DNA. NER operates throughout the genome, but the efficiencies of lesion removal are not the same for different genomic regions. Even within a single gene or DNA strand repair rates vary, and this intragenic heterogeneity is of considerable interest with respect to the mutagenic potential of carcinogens. In this study, we have analyzed the removal of the two major types of genotoxic DNA adducts induced by UV light, i.e., the pyrimidine (6-4)-pyrimidone photoproduct (6-4PP) and the cyclobutane pyrimidine dimer (CPD), from the Saccharomyces cerevisiae URA3 gene at nucleotide resolution. In contrast to the fast and uniform removal of CPDs from the transcribed strand, removal of lesions from the nontranscribed strand is generally less efficient and is modulated by the chromatin environment of the damage. Removal of 6-4PPs from nontranscribed sequences is also profoundly influenced by positioned nucleosomes, but this type of lesion is repaired at a much higher rate. Still, the transcribed strand is repaired preferentially, indicating that, as in the removal of CPDs, transcription-coupled repair predominates in the removal of 6-4PPs from transcribed DNA. The hypothesis that transcription machinery operates as the rate-determining damage recognition entity in transcription-coupled repair is supported by the observation that this pathway removes both types of UV photoproducts at equal rates without being profoundly influenced by the sequence or chromatin context.

FIG. 1

Enzymatic detection of UV-induced photoproducts at nucleotide resolution. DNA (25 μg) isolated from cells exposed to either 0 (lanes 1 and 4) or 140 J/m² (lanes 2, 3, 5, and 6–9) was mock treated (lanes 2, 5, 7, and 9) or treated with photoreactivating enzyme (PRE) (lanes 3, 6, and 8) and subsequently treated with either T4 endonuclease V (lanes 1–3 and 7) or UVDE (lanes 4–6, 8, and 9). Lanes 2 and 7 show CPD-specific incision, and lanes 6 and 8 show 6-4PP-specific incision, while in lanes 5 and 9 the combined distribution pattern is observed. Irr., irradiation; T4, phage enzyme T4 endonuclease V.

FIG. 2

Repair of UV-induced CPDs at single nucleotide resolution along (A) the transcribed strand, nt 268 to 607 (all positions are relative to the start codon, ATG, designated +1), and (B) the nontranscribed strand, nt −151 to 221, nt 324 to 518, and nt 478 to 760. Cells were irradiated with 70 J/m², and repair was monitored at 0, 40, 80, and 120 min after irradiation. Samples that were mock treated or treated with the CPD-specific enzyme T4endoV are denoted by − and +, respectively. Shaded boxes indicate the internal protected regions of nucleosomes U1, U2, U4, and U5 positioned along the URA3 locus (19). Dark arrows mark CPDs that persisted after 2 h of repair, and open arrows mark some positions that were repaired very fast. (C) Graphic representation of quantified CPD repair rates along the nontranscribed strand of the URA3 locus. Repair t_1/2 values, determined as the time at which 50% of the initial CPD signal was removed, were calculated for each individual CPD position with a sufficient signal-to-noise ratio and are plotted above their corresponding dipyrimidine positions. The internal protected regions are represented by the shaded boxes inside nucleosomes U1 through U6 (19).

FIG. 3

(A) Repair of UV-induced 6-4PPs along the nontranscribed strand of the URA3 gene. Numbering of arrows is as follows: 1, 5′-TC-3′ (nt −2 and −1; 2, 5′-TC-3′ (nt 4 and 5); 3, 5′-CTTC-3′ (nt 101 to 104); 4, 5′-TCCC-3′ (nt 156 to 159); 5, 5′-CT-3′ (nt 172 and 173); and 6, 5′-TTCC (nt 204 to 207). (B) Repair-proficient (RAD⁺) cells are compared with isogenic rad7 mutant cells. Cells were irradiated with 140 J/m², and repair was monitored at 0, 20, 40, and 60 min after irradiation. To account for non-dimer-specific incision nonirradiated DNA was also assayed (indicated by a minus sign). Shaded boxes indicate the internal protected regions of nucleosomes U1, U2, and U5 positioned along the URA3 locus (19).

FIG. 4

Repair of UV-induced 6-4PPs along the URA3 transcribed strand in repair-proficient (RAD⁺) cells (A) and in isogenic rad7 mutant cells (B). Cells were irradiated with 140 J/m², and repair was monitored at 0, 20, 40, and 60 min. The minus signs indicate nonirradiated DNA assayed with UVDE. Several strong UV-independent incision products appearing at nondinucleotide sequences are indicated (asterisks); these were left out of the analysis. Arrows point to positions where the repair rate is elevated with respect to the general repair rate.

FIG. 5

Repair of UV-induced CPDs (A) and 6-4PPs (B) along the URA3 template strand in a rad7 strain. Repair was monitored at 0, 20, 40, and 60 min after irradiation (70 J/m²). The large arrow indicates the major transcription-initiation site (+1) and the direction of transcription. To account for background levels, nonirradiated DNA was also assayed (indicated by a minus sign). Due to the lower induction frequency of 6-4PPs than of CPDs, twice the amount of DNA was assayed in the 6-4PP analysis and different exposure times were used to allow visual inspection. As calculated from short exposures of the undamaged full-length fragment (not indicated), the autoradiograms display a 3- to 3.5-fold amplification of the actual 6-4PP signal relative to that of the CPDs. The asterisk indicates a UV-independent background signal.