Abstract
Transcription-coupled DNA repair is dedicated to the removal of DNA lesions from transcribed strands of expressed genes. RNA polymerase arrest at a lesion has been proposed as a sensitive signal for recruitment of repair enzymes to the lesion site. To understand how initiation of transcription-coupled repair may occur, we have characterized the properties of the transcription complex when it encounters a lesion in its path. Here we have compared the effect of cisplatin-induced intrastrand cross-links on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We found that a single cisplatin 1,2-d(GG) intrastrand cross-link or a single cisplatin 1,3-d(GTG) intrastrand cross-link is a strong block to both polymerases. Furthermore, the efficiency of the block at a cisplatin 1,2-d(GG) intrastrand cross-link was similar in several different nucleotide sequence contexts. Interestingly, some blockage was also observed when the single cisplatin 1,3-d(GTG) intrastrand cross-link was located in the non-transcribed strand. Transcription complexes arrested at the cisplatin adducts were substrates for the transcript cleavage reaction mediated by the elongation factor TFIIS, indicating that the RNA polymerase II complexes arrested at these lesions are not released from template DNA. Addition of TFIIS yielded a population of transcripts up to 30 nucleotides shorter than those arrested at the lesion. In the presence of nucleoside triphosphates, these shortened transcripts could be re-elongated up to the site of the lesion, indicating that the arrested complexes are stable and competent to resume elongation. These results show that cisplatin-induced lesions in the transcribed DNA strand constitute a strong physical barrier to RNA polymerase progression, and they support current models of transcription arrest and initiation of transcription-coupled repair.
Highlights
A current model for Transcription-coupled repair (TCR) proposes that RNA polymerase arrested at a lesion in DNA constitutes a signal for the repair proteins to initiate repair
In the presence of nucleoside triphosphates, these shortened transcripts could be re-elongated up to the site of the lesion, indicating that the arrested complexes are stable and competent to resume elongation. These results show that cisplatin-induced lesions in the transcribed DNA strand constitute a strong physical barrier to RNA polymerase progression, and they support current models of transcription arrest and initiation of transcription-coupled repair
To study the effect of a single cis 1,2-d(GG) or a single cis 1,3-d(GTG) on transcription, we have developed an in vitro transcription system consisting of DNA substrates containing a single cis 1,2-d(GG) in two different sequence contexts and a single cis 1,3-d(GTG) located in the transcribed or the nontranscribed strand downstream of the T7 promoter or the adenovirus major late promoter (AdMLP), with purified T7 RNA polymerase (RNAP) or rat liver RNAPII and initiation factors, respectively
Summary
Genomic nucleotide excision repair and by TCR [22,23,24] These adducts may impose a more serious problem for an elongating RNA polymerase compared with a CPD, because they cause substantial unwinding and bending of the DNA helix (reviewed in Ref. 21). These lesions have been shown to block transcription by T3, E. coli, and wheat germ RNAP [25,26,27]. The arrested RNAPII complex was stable, as indicated by the ability of elongation factor TFIIS to induce transcript cleavage, producing a population of transcripts up to 30 nts shorter than those arrested at the lesion, which could be re-elongated up to the lesion when the nucleoside triphosphate precursors were added
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