Nucleotide Sequence Context Effect of a Cyclobutane Pyrimidine Dimer upon RNA Polymerase II Transcription

Abstract

We have studied the role of sequence context upon RNA polymerase II arrest by a cyclobutane pyrimidine dimer using an in vitro transcription system consisting of templates containing a specifically located cyclobutane pyrimidine dimer (CPD) and purified RNA polymerase II (RNAP II) and initiation factors. We selected a model sequence containing a well characterized site for RNAP II arrest in vitro, the human histone H3.3 gene arrest site. The 13-base pair core of the arrest sequence contains two runs of T in the nontranscribed strand that impose a bend in the DNA. We hypothesized that arrest of RNAP II might be affected by the presence of a CPD, based upon the observation that a CPD located at the center of a dA 6 ⅐dT 6 tract eliminates bending (Wang, C.-I., and Taylor, J.-S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9072-9076). We examined the normal H3.3 sequence and a mutant sequence containing a T 3 G transversion, which reduces bending and efficiency of arrest. We show that a CPD in the transcribed strand at either of two locations in the arrest site is a potent block to transcription. However, a CPD in the nontranscribed strand only transiently pauses RNAP II. The CPD in concert with a mutation in the arrest site can reduce the extent of bending of the DNA and improve readthrough efficiency. These results demonstrate the potential importance of sequence context for the effect of CPDs within transcribed sequences.DNA repair rates along the genome are clearly heterogeneous (2). Certain lesions in actively transcribed genes are repaired much faster than in inactive loci. This can be generally ascribed to the preferential repair of the transcribed strand over the nontranscribed strand (3). Transcription-dependent repair has been shown to occur in mammalian cells (4, 5), in Escherichia coli (6), and in Saccharomyces cerevisiae (7-9), suggesting that it is a widespread phenomenon reflecting the existence of a mechanism that directly couples nucleotide excision repair and transcription.An additional level of repair heterogeneity is represented by the sequence-specific rates of repair of UV-induced cyclobutane pyrimidine dimers (CPDs) 1 (10 -13). Analysis of repair of CPDs along the tumor suppressor gene p53 (11) revealed that the sites at which CPDs were poorly repaired were also hot spots for mutations found in skin cancers, demonstrating a direct correlation between slow repair of CPDs and mutation hot spots in skin cancer. A correlation between slow repair rates and occurrence of mutations has also been observed in the hypoxanthine-guanine phosphoribosyltransferase gene in human fibroblasts treated with benzo[a]pyrene diol epoxide (14). Slow repair of CPDs at particular sites on the transcribed strand (10, 11) may reflect efficient bypass of CPDs by RNA polymerase II (RNAP II) in specific sequence contexts. A dimer might even be masked in particular sequence contexts such that transcription-coupled repair would not be initiated.The role of the sequence context of a lesion on transcription can be analyzed more ...

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

“…The undamaged control template and the one with a single CPD in the nontranscribed strand behaved in the same way (Fig. 5), confirming previous reports that a CPD in the nontranscribed strand does not affect the behavior of a transcribing RNAP II (10), unless it has an effect on the inherent structure of the DNA helix (16). In this case, when a thymine dimer was introduced into the nontranscribed strand of a naturally strong RNAP II transcription arrest site, it caused the arrest site to become weaker and only transiently arrested the polymerase.…”

“…Its atomic structure has been solved at a 2.8 Å resolution (36). Lesions that have been shown to block an elongating RNAP II in vitro when present on the transcribed strand include cisplatin GG intrastrand and interstrand cross-link (37)(38)(39), N-acetoxyacetylaminofluorene (40), psoralen monoadducts and interstrand cross-links (41), benzo(a)pyrene (42), and CPDs (10,16,17,43).…”

“…11). Depending upon the sequence context, however, they can affect the rate of elongation or stalling of the polymerase (16). In this study we examined the behavior of T7 RNAP and mammalian RNAP II when transcribing a template that contains two closely spaced lesions, one on each strand, to investigate their possible combinatorial effect upon transcription elongation.…”

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

“…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 initiation factors. Using this in vitro transcription system, we have previously shown that a cyclobutane pyrimidine dimer in the transcribed strand in different sequence contexts is a strong block to RNAP II (9,10). The arrested polymerase is stable and competent to resume transcription.…”

Post-Transcriptional Regulation of DNA Damage-Responsive Gene Expression

“…These DNA lesions alter DNA structure and pose a block to elongating RNA polymerase II complexes; so, UV light leads to a dose-dependent decrease in the synthesis of nascent mRNA (Fig. 1A) (69,101,106,136,138,149). The probability that the synthesis of a specific mRNA is blocked is a function of UV dose and the length of the gene (136).…”