Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template.

Abstract

A current model for transcription-coupled DNA repair is that RNA polymerase, arrested at a DNA lesion, directs the repair machinery to the transcribed strand of an active gene. To help elucidate this role of RNA polymerase, we constructed DNA templates containing the major late promoter of adenovirus and a cyclobutane pyrimidine dimer (CPD) at a specific site. CPDs, the predominant DNA lesions formed by ultraviolet radiation, are good substrates for transcriptioncoupled repair. A CPD located on the transcribed strand of the template was a strong block to polymerase movement, whereas a CPD located on the nontranscribed strand had no effect on transcription. Furthermore, the arrested polymerase shielded the CPD from recognition by photolyase, a bacterial DNA repair protein. Transcription elongation factor SIU (also called TFIIS) facilitates read-through of a variety of transcriptional pause sites by a process in which RNA polymerase II cleaves the nascent transcript before elongation resumes. We show that SU1 induces nascent transcript cleavage by RNA polymerase H stalled at a CPD. However, this cleavage does not remove the arrested polymerase from the site of the DNA lesion, nor does it facilitate translesional bypass by the polymerase. The arrested ternary complex is stable and competent to resume elongation, demonstrating that neither the polymerase nor the RNA product dissociates from the DNA template.Helix-distorting lesions are produced in cellular DNA by various endogenous and exogenous agents. Among such lesions is the cyclobutane pyrimidine dimer (CPD), the most prevalent lesion formed by short-wavelength UV radiation. CPDs can block DNA replication and transcription, leading to cell death. If unrepaired, the DNA damage can lead to mutagenesis, activation of protooncogenes, and ultimately carcinogenesis. One mechanism to remove these lesions is nucleotide excision repair (NER), common to a wide range of species from Escherichia coli to humans. The basic mechanism of NER is well understood in E. coli (1). Recognition of the damage is followed by incision of the damaged DNA strand on both sides of the lesion. The damage-containing oligonucleotide is removed, the resultant gap is filled in by DNA polymerase, and DNA ligase completes the process by joining the repair patch to the contiguous DNA strand. Although the detailed mechanism of NER in eukaryotes is not established as firmly, it appears to have the same essential features. A striking property of NER is the intragenomic heterogeneity of repair efficiency (2). Expressed genes are repaired more rapidly than the overall genome in rodent (3) and human (4) cells in culture. Furthermore, this preferential repair is largely due to efficient repair of the transcribed strand of an active gene compared to the nontranscribed strand or unexpressed DNA sequences (5). In addition to mammalian cells, preferential repair of transcribed DNA strands has been demonstrated in E. coli (6) and Saccharomyces cerevisiae (7-9), so it is likely to be a universal phenome...

p21-induced cycle arrest in G1 protects cells from apoptosis induced by UV-irradiation or RNA polymerase II blockage

“…At doses of UVB which impaired the induction of p21 Waf1/Cip1 protein expression (approximately 500 J/m 2 and over), apoptosis was induced. It has been shown that UV radiation transciently inhibits both DNA and RNA synthesis in vivo (Mayne and Lehmann, 1982) and completely blocks RNA pol II transcription in vitro (Donahue et al, 1994;Selby et al, 1997). Furthermore, it has been suggested previously that blockage of RNA polymerase may trigger UV-induced apoptosis (Ljungman and Zhang, 1996).…”

“…For example, 6 and 12 h incubations with mimosine elevate p21 Waf1/Cip1 levels but the levels decline after UV-irradiation ( Figure 9); however, a 24 h incubation with mimosine results in the maintenance of high levels of p21 Waf1/Cip1 following irradiation. These post-irradiation di erences in p21 Waf1/Cip1 levels must re¯ect di erences in the relative rates of translation and proteolysis since transcription is blocked at the dose of UVB used (2000 J/m 2 ) (Mayne and Lehmann, 1982;Donahue et al, 1994;Selby et al, 1997). Both p21 Waf1/Cip1 and p53 protein degradation proceed via ubiquitination (Maki et al, 1996;Maki and Howley, 1997) and p53 stabilization involves reduced ubiquitination (Maki and Howley, 1997).…”

Cockayne syndrome group B protein enhances elongation by RNA polymerase II

“…Effect of CSB on RNAPII Stalled by DNA Damage. Elongation by RNAPII is known to be completely blocked by a TϽϾT in the template strand (24). To examine a possible role of CSB on the blocked polymerase we constructed pPU192, which possesses a TϽϾT located in the template strand at positions 149-150 downstream from the adenovirus major late promoter transcription start site (14).…”

p53-mediated transcription induces resistance of DNA to UV inactivation

“…One possibility is that the transcription machinery reads or skips over regions containing thymine dimers in the DNA. We consider this very unlikely because it has been demonstrated that a cyclobutane pyrimidine dimer located on the template strand, but not on the nontranscribed strand, blocks transcriptional elongation and arrests the RNA polymerase at the site of the lesion (Protic-Sabljic and Kraemer, 1985;Sauerbier and Hercules, 1978;Selby and Sancer, 1990;Donahue et al, 1994). At best, this would result in the production of truncated and therefore inactive CAT enzyme.…”