Abstract
Transcription and repair of many DNA helix-distorting lesions such as cyclobutane pyrimidine dimers have been shown to be coupled in cells across phyla from bacteria to humans. The signal for transcription-coupled repair appears to be a stalled transcription complex at the lesion site. To determine whether oxidative DNA lesions can block correctly initiated human RNA polymerase II, we examined the effect of site-specifically introduced oxidative damages on transcription in HeLa cell nuclear extracts. We found that transcription was blocked by single-stranded breaks, common oxidative DNA lesions, when present in the transcribed strand of the transcription template. Cyclobutane pyrimidine dimers, which have been previously shown to block transcription both in vitro and in vivo, also blocked transcription in the HeLa cell nuclear transcription assay. In contrast, the oxidative DNA base lesions, 8-oxoguanine, 5-hydroxycytosine, and thymine glycol did not inhibit transcription, although pausing was observed with the thymine glycol lesion. Thus, DNA strand breaks but not oxidative DNA base damages blocked transcription by RNA polymerase II.
Highlights
Transcription-coupled repair (TCR)1 is a specialized form of DNA repair where damages are repaired preferentially in the transcribed strand of actively transcribed genes
RNA Polymerase II Transcription Is Blocked by Site-specific Single-stranded Breaks in the Template Strand—To determine what effect single-stranded breaks (SSBs) had on transcription by RNA polymerase II, we carried out run-off transcription assays with HeLa cell nuclear extracts on templates containing SSBs with different end chemistries located on the transcribed strand downstream from the HIV-1 promoter
The number of radioactive nucleotides incorporated into the transcript was taken into account, since the difference in the number of radioactive nucleotides incorporated between the run-off transcript and a transcript blocked at the lesion was significant (ϳ7% in the case of the 54-mer damage-containing oligonucleotides and ϳ9% in the case of the 71-mer damage-containing oligonucleotides)
Summary
Introduction of Unique Damages to Transcription Templates—The damages in the transcription templates were located exclusively on the transcribed strand throughout this study. The furan-containing oligonucleotide (Microbiology and Molecular Genetics DNA Synthesis Facility, University of Vermont) (5Ј-AAGCTTGGCACTGFuranCCGTCGTTTTACAACGTCGTG-3Ј) annealed to its complement, was incubated with 36.4 ng of HeLa cell nuclear extract in 45 mM HEPES (pH 7.9), 70 mM KCl, 1 mM DTT, 2 mM EDTA, and 5 mM MgCl2. The 8-oxoG-containing oligonucleotide (Microbiology and Molecular Genetics DNA Synthesis Facility, University of Vermont) (5Ј-AAGCTTGGCACTGGCC8-oxoGTCGTTTTACAACGTCGTG-3Ј) annealed to its complement, was incubated with 3.64 g of HeLa cell nuclear extract in 25 mM HEPES (pH 7.9), 50 mM KCl, 2 mM DTT, 2 mM EDTA, and 2.5% glycerol. To assay for the presence of the lesion, 2.5 fmol of template/substrate was incubated with 60 fmol of the appropriate enzyme for 60 min at 37 °C in 10 mM Tris (pH 8), 1 mM EDTA, 50 mM NaCl. The oligonucleotide containing the CPD dimer was end-labeled with [␥-32P]ATP by T4 polynucleotide kinase and annealed to its complementary strand. The results were visualized and quantified by autoradiography and/or analysis on an isotope imaging system (Molecular Images FX System, Bio-Rad)
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