Transcription Arrest at a Lesion in the Transcribed DNA Strand in Vitro Is Not Affected by a Nearby Lesion in the Opposite Strand

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 transcriptioncoupled 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.It has been nearly two decades since the discovery that in UV-irradiated cells of eukaryotes and prokaryotes the transcribed strand of an active gene is repaired more rapidly than the nontranscribed strand (1, 2). However, the mechanistic details of this phenomenon, termed transcription-coupled repair (TCR), 1 are still not fully understood. The general model for the mechanism of TCR postulates that an RNA polymerase stalled at a lesion is the signal for recruitment of the repair proteins and initiation of excision repair (3). In the case of UV-induced DNA lesions, such as the most frequently formed cis-syn 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 finally the repair patch is ligated to the contiguous DNA (reviewed in Refs. 4 and 5). An active RNA polymerase is a prerequisite for TCR (6 -8). However, the arrested polymerase must then be displaced so that the lesion becomes accessible to the repair machinery (3). In Escherichia coli, a transcription-repair coupling factor (Mfd) has been shown to displace the stalled polymerase and facilitate TCR (9). In eukaryotes, however, it is still not clear how the arrested polymerase signals the assembly of the repair machinery at the site of damage, nor is it clear how the polymerase is displaced to allow access of the repair proteins to the damaged site. Despite the evidence that incomple...

Behavior of T7 RNA Polymerase and Mammalian RNA Polymerase II at Site-specific Cisplatin Adducts in the Template DNA

“…Similarly, T7 RNAP can bypass several bulky lesions including CPD (19,49), acetylaminofluorene (50), psoralen adducts (33), and BPDE adducts (51) more readily than RNAPII. Perlow et al (48) have proposed that the ability of T7 RNAP to readthrough an anti-BPDE DNA adduct is due to the more open structure of the catalytic site of the T7 enzyme compared with that of the eukaryotic RNAPII, as revealed from the crystal structures of these proteins (52)(53)(54).…”

Fate of RNA Polymerase II Stalled at a Cisplatin Lesion

“…Transcription complexes stalled at a site of UV DNA damage such as thymine dimers has been widely studied (13)(14)(15). Studies presented here focus on another type of lesion repaired by NER/TCR: cisplatin cross-links (16,17).…”

Role of DNA repair in the protection against genotoxic stress

“…GGR takes place throughout the genome, irrespective of whether the target sequence is silent or actively transcribed, and this XPCdependent process constitutes an important mechanism for the removal of potentially mutagenic DNA lesions that ultimately lead to cancer [34,35,162]. However, unrepaired DNA lesions not only cause mutations but also interfere with the transcriptional process, thus perturbing normal gene functions and triggering cell senescence or apoptosis [162,163]. Therefore, living organisms have developed the fast transcription-coupled repair (TCR) subpathway to remove DNA lesions from the transcribed strand of active genes [164].…”