Single-Stranded Breaks in DNA but Not Oxidative DNA Base Damages Block Transcriptional Elongation by RNA Polymerase II in HeLa Cell Nuclear Extracts

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.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 (for reviews, see Refs. 1 and 2). TCR was originally believed to be a subpathway of nucleotide excision repair; however, ionizing radiation damage (3), 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol; Tg) (4, 5), and 7,8-dihydro-8-oxoguanine (8-oxoG) (5) are removed in a TCR-dependent manner from human cells that lack nucleotide excision repair. Since Tg and 8-oxoG are small nonbulky lesions that are repaired primarily by the base excision repair (BER) pathway (for reviews, see Refs. 6 -8), TCR of Tg and 8-oxoG in cells that lack nucleotide excision repair (5) links TCR to BER. TCR of 8-oxoG has also been shown to occur in nonreplicating Escherichia coli cells (9). TCR of oxidative damage does not appear to be universal, since in Chinese hamster ovary cells, TCR of oxidative damage produced by photosensitization and oxidizing agents is not observed in the Dhfr and cFos genes (10 -12). Furthermore, DNA strand breaks, oxidative lesions also repaired by BER, do not appear to be repaired by TCR in the Dhfr gene from Chinese hamster ovary cells (13) or human colon cancer cells (14). Interestingly, recent measurements of TCR of cyclobutane pyrimidine dimers in the Hprt gene, integrated at different sites in Chinese hamster ovary cell chromosomes, have suggested that preferential repair of actively transcribed genes, as well as preferential repair of damages in the transcribed strand, is significantly affected by genomic context (15).The proposed signal for TCR is an RNA polymerase transcription complex stalled at a lesion, which recruits the repair proteins to the damage site (16 -18); the ability of a lesion on the transcribed strand to block the RNA polymerase transcription complex has been assumed to be crucial for T...

The role of Cockayne Syndrome group B (CSB) protein in base excision repair and aging

“…8-oxoG, thymine glycol) to block RNA pol II during transcription. Some studies show that RNA pol II can bypass an 8-oxoG lesion (Kathe et al, 2004;Larsen et al, 2004;Tornaletti et al, 2004;Viswanathan and Doetsch, 1998), which is a non-bulky lesion repaired by BER, while some other groups could see a partial stalling of RNA pol II . Sarasin and coworkers have suggested that the repair of 8-oxoG on a transcribed strand results in a competition between BER and TCR components, dependent upon which enzymes arrive first to the lesion (Pastoriza-Gallego et al, 2007).…”

Clinical implications of the basic defects in Cockayne syndrome and xeroderma pigmentosum and the DNA lesions responsible for cancer, neurodegeneration and aging

“…Therefore a unifying principle for damage-dependent neurodegeneration would be the prediction that deficits in TCR and SSB repair should lead to increased apoptosis of post-mitotic neurons, especially since single strand breaks are also transcription blocking lesions (Kathe et al, 2004). Neurodegeneration might be ascribed to the greater relative importance of a TCR-like repair in differentiated brain cells such that a failure of TCR triggers an apoptotic response (Ljungman and Zhang, 1996;Proietti De Santis et al, 2002;D'Errico et al, 2003).…”

“…A recent suggestion that RNA pol II is a universal sensor of DNA damage (Lindsey-Boltz and Sancar, 2007) blurs the distinction between TCR and global repair. The neurological symptoms of CS patients have been ascribed to defective repair in the brain of endogenous oxidative damage that blocks transcription (Kuraoka et al, 2000;Osterod et al, 2002;de Waard et al, 2003;Kyng et al, 2003;Tuo et al, 2003;Cline et al, 2004), but the more common oxidative base damages (8-oxo-G, 5-hydroxycytosine, thymine glycols) do not block transcription and are therefore not the culprits in neurodegeneration (Kathe et al, 2004). CSA and CSB cells are however different in their responses to oxidative damage, despite overlap in clinical symptoms (de Waard et al, 2004;D'Errico et al, 2007).…”

“…The oxidative lesion 8-oxo-G does not appear to accumulate in CS autopsy material (Hayashi et al, 2005) despite the higher amounts of protein oxidation and lipid oxidation in the brains of CS patients (Hayashi et al, 2001) and crossing Cs-b mice with Ogg1 deficient mice did not enhance the neurological symptoms (Laposa et al, 2007b). Surprising is the absence of reported neurological or aging phenotypes among mouse strains lacking glycosylases which are the recognition and excision enzymes for BER that would be expected to show increased sensitivity to ROS (Friedberg et al, 1998), although many oxidative base damages are not transcription blocking lesions (Kathe et al, 2004). Perhaps these strains should be screened more carefully, or combined strains made lacking several glycosylases to increase the chances of detecting such a phenotype.…”

“…Lipid oxidation products produce malondialdehydedeoxyguanosine (M-1-dG) adducts in DNA that block transcription (Cline et al, 2004). Analysis in a cell-free system showed that although UV-induced pyrimidine dimers and single strand breaks (SSBs) were effective blocks to transcription, base damage of the kind subject to BER were not (Kathe et al, 2004).…”