Abstract
Cis-syn cyclobutane pyrimidine dimers (CPDs) are the most frequently formed lesions in UV-irradiated DNA. CPDs are repaired by the nucleotide excision repair pathway. Additionally, they are subject to transcription-coupled DNA repair. In the general model for transcription-coupled DNA repair, an RNA polymerase arrested at a lesion on the transcribed DNA strand facilitates repair by recruiting the repair machinery to the site of the lesion. Consistent with this model, transcription experiments in vitro have shown that CPDs in the transcribed DNA strand interfere with the translocation of prokaryotic and eukaryotic RNA polymerases. Here, we study the behavior of RNA polymerase when transcribing a template that contains two closely spaced lesions, one on each DNA strand. Similar DNA templates containing no CPD, or a single CPD on either the transcribed or the nontranscribed strand were used as controls. Using an in vitro transcription system with purified T7 RNA polymerase (T7 RNAP) or rat liver RNAP II, we characterized transcript length and efficiency of transcription in vitro. We also tested the sensitivity of the arrested RNAP II-DNA-RNA ternary complex, at a CPD in the transcribed strand, to transcription factor TFIIS. The presence of a nearby CPD in the nontranscribed strand did not affect the behavior of either RNA polymerase nor did it affect the reverse translocation ability of the RNAP II-arrested complex. Our results additionally indicate that the sequence context of a CPD affects the efficiency of T7 RNAP arrest more significantly than that of RNAP II.
Highlights
UV-induced DNA lesions, such as the most frequently formed cis-syn cyclobutane pyrimidine dimers (CPDs), the specific repair process requires that an oligonucleotide containing the damaged site is excised from the DNA, the gap created is closed by repair replication, and the repair patch is ligated to the contiguous DNA
We found that when two lesions were present in the DNA template, transcription arrest of both T7 RNA polymerase (T7 RNAP) and RNAP II occurred at the same location as in the template with a single CPD in the transcribed strand, indicating that the CPD in the nontranscribed strand had no effect on the response of either polymerase
The presence of a CPD in the nontranscribed strand had no effect on transcription by T7 RNAP and resulted in transcripts that were 112 nucleotides long (Fig. 2, lane 3)
Summary
Vol 278, No 21, Issue of May 23, pp. 19558 –19564, 2003 Printed in U.S.A. Transcription Arrest at a Lesion in the Transcribed DNA Strand in Vitro Is Not Affected by a Nearby Lesion in the Opposite Strand*. In vitro transcription systems with DNA templates containing different types of lesions have proved very useful for demonstrating that several types of lesions in the transcribed DNA strand can block transcription by different RNA polymerases (reviewed in Ref. 11) These findings are in agreement with the proposed model for TCR that requires an arrested RNA polymerase to signal the recruitment of the repair enzymes to the damaged site [14]. It was demonstrated previously that when RNAP II from rat liver is stalled at a CPD in vitro, addition of TFIIS caused cleavage of the nascent transcript and reverse translocation of the polymerase, up to 35 nucleotides upstream of the lesion without disruption of the ternary complex [17] This facilitated repair of the CPD when a small repair enzyme, photolyase, was added into the reaction; subsequent resumption of transcription past the repaired site was documented. We tested the effect of TFIIS on the behavior of RNAP II by transcribing the template with the two closely opposed lesions and observed the same cleavage pattern with the template carrying a single CPD on the transcribed strand
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