Effect of Thymine Glycol on Transcription Elongation by T7 RNA Polymerase and Mammalian RNA Polymerase II

Abstract

Thymine glycols are formed in DNA by exposure to ionizing radiation or oxidative stress. Although these lesions are repaired by the base excision repair pathway, they have been shown also to be subject to transcription-coupled repair. A current model for transcription-coupled repair proposes that RNA polymerase II arrested at a DNA lesion provides a signal for recruitment of the repair enzymes to the lesion site. Here we report the effect of thymine glycol on transcription elongation by T7 RNA polymerase and RNA polymerase II from rat liver. DNA substrates containing a single thymine glycol located either in the transcribed or nontranscribed strand were used to carry out in vitro transcription. We found that thymine glycol in the transcribed strand blocked transcription elongation by T7 RNA polymerase ϳ50% of the time but did not block RNA polymerase II. Thymine glycol in the nontranscribed strand did not affect transcription by either polymerase. These results suggest that arrest of RNA polymerase elongation by thymine glycol is not necessary for transcription-coupled repair of this lesion. Additional factors that recognize and bind thymine glycol in DNA may be required to ensure RNA polymerase arrest and the initiation of transcription-coupled repair in vivo. Transcription-coupled repair (TCR)1 is a pathway of DNA excision repair that is targeted to removal of DNA lesions present in transcribed strands of expressed genes (1). TCR has been demonstrated in mammalian cells (2), Escherichia coli (3), and Saccharomyces cerevisiae (4 -6). TCR was originally observed for lesions repaired by nucleotide excision repair (2). An active RNA polymerase elongation complex is necessary for preferential repair of the transcribed strand (7). The arrest of transcription at DNA lesions has been proposed to serve as a specific signal to direct repair enzymes to the transcribed strand of an active gene to initiate a repair event (8). It is likely that this repair pathway evolved for the dedicated purpose of resolving the impasse of an RNA polymerase arrested at a lesion. The polymerase must be displaced both to permit verification that a lesion caused the arrest and to allow the repair enzymes to operate on the lesion (1).The role of RNA polymerases in TCR has been examined more directly by comparing the extent of RNA polymerase arrest in vitro by a lesion with TCR of that lesion in vivo. These studies have shown that various types of DNA damage in the template strand of DNA can act as blocks to transcription catalyzed by different RNA polymerases. In several cases, a correlation between the extent of polymerase arrest and the extent of TCR has been found (7). However, most of these studies have been carried out using viral and bacterial RNA polymerases. In an attempt to understand the role of mammalian RNA polymerase II (RNAP II) in TCR, we have developed an in vitro transcription system with templates containing a site-specific lesion positioned downstream of the adenovirus major late promoter (AdMLP) and purified proteins and i...

Pathogen DNA as target for host-generated oxidative stress: Role for repair of bacterial DNA damage in Helicobacter pylori colonization

“…Some of the free-radical-modified DNA bases are not mutagenic but can block RNA and DNA polymerases (14,15). Consequently, HpNth could provide H. pylori with a defense against the lethal effect of exogenous oxidizing agents.…”

“…In contrast, pyrimidine residues are chemically more resistant, but some of their oxidation products, such as 5,6-dihydroxydihydrothymine [known as thymine glycol], can entail lethal consequences for the cell (13). This lesion can block both DNA and RNA polymerases (14,15). Other common pyrimidine derivatives are 5,6-dihydrothymine and urea (13).…”

Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II

“…Oxidized and alkylated bases, produced during normal cellular metabolism, are generally cleared from DNA by base excision repair (9,10). Whereas TCR of oxidative damage in eukaryotic cells has been suggested (11), neither 7,8-dihydro-8-oxoguanine nor thymine glycol significantly affects translocation of RNAPII in vitro (12,13). Therefore, a role for TCR in the repair of endogenous oxidative DNA damage remains to be clearly established.…”

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

“…In many cases, the effects of oxidative DNA damages on transcription by RNA polymerases differ from their effects on DNA polymerases. Tg is a block to T7 RNA polymerase (23)(24)(25). However, RNA polymerase II, partially purified from rat liver, completely bypassed Tg lesions in the transcribed strand located downstream from the adenovirus major late promoter, and the addition of fractions containing transcription factor II D and transcription factor II H (TFIIH) did not have any measurable effect (24).…”

“…Tg is a block to T7 RNA polymerase (23)(24)(25). However, RNA polymerase II, partially purified from rat liver, completely bypassed Tg lesions in the transcribed strand located downstream from the adenovirus major late promoter, and the addition of fractions containing transcription factor II D and transcription factor II H (TFIIH) did not have any measurable effect (24). As with DNA polymerases, 8-oxoG does not block T7 (23,25) or E. coli RNA polymerase in vitro (26) or in cells (9) and at best stalls RNA polymerase II in vitro with a template containing a poly(C) tail (27).…”