Cyclobutane Pyrimidine Dimers and Bulky Chemical DNA Adducts Are Efficiently Repaired in Both Strands of Either a Transcriptionally Active or Promoter-deleted APRT Gene

Yi Zheng,Gerald M. Adair,Annie Pao,Moon-shong Tang

doi:10.1074/jbc.m010973200

Abstract

Both prokaryotic and eukaryotic cells have the capacity to repair DNA damage preferentially in the transcribed strand of actively expressed genes. However, we have found that several types of DNA damage, including cyclobutane pyrimidine dimers (CPDs) are repaired with equal efficiency in both the transcribed and nontranscribed strands of the adenine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells. We further found that, in two mutant cell lines in which the entire APRT promoter region has been deleted, CPDs are still efficiently repaired in both strands of the promoterless APRT gene, even though neither strand appears to be transcribed. These results suggest that efficient repair of both strands at this locus does not require transcription of the APRT gene. We have also mapped CPD repair in exon 3 of the APRT gene in each cell line at single nucleotide resolution. Again, we found similar rates of CPD repair in both strands of the APRT gene domain in both APRT promoter-deletion mutants and their parental cell line. Our findings suggest that current models of transcription-coupled repair and global genomic repair may underestimate the importance of factors other than transcription in governing the efficiency of nucleotide excision repair.

Highlights

Both prokaryotic and eukaryotic cells have the capacity to repair DNA damage preferentially in the transcribed strand of actively expressed genes
Nucleotide excision repair (NER) is thought to involve two distinct subpathways: transcriptioncoupled repair (TCR), which selectively and very efficiently repairs transcription-blocking damage in the transcribed strand of actively expressed genes, and global genomic repair (GGR), which is responsible for repairing damage in the nontranscribed strand and the rest of the genome [2, 5,6,7,8]
In contrast to the preferential repair of cyclobutane pyrimidine dimers (CPDs) in the transcribed strand, which is observed in the Chinese hamster ovary (CHO) dihydrofolate reductase (DHFR) gene, we have found that CPDs on both strands of the CHO adenine phosphoribosyltransferase (APRT) gene are repaired with equal efficiency

Summary

EXPERIMENTAL PROCEDURES

Cell Culture, and Carcinogen Treatment—The Chinese hamster CHO-AT3-2 cell line is hemizygous for the endogenous APRT gene locus [20]; these cells contain a single, actively transcribed APRT gene, which is located on the CHO Z7 chromosome. Strand-specific DNA Probes—pGEM-zf11(ϩ)-APRT and pGEMzf11(Ϫ)-APRT vectors were constructed by inserting a 3.9-kb BamHI fragment containing the CHO APRT gene into the pGEM-zf11(ϩ) or (Ϫ) vectors These two constructs allowed us to isolate either the transcribed (T) or the nontranscribed (NT) strand of the APRT gene. To generate 32P-labeled DNA probes for either the T or NT strand, 0.1 ␮g of template DNA was added to reaction buffer containing 50 pmol of the appropriate strand-specific primer, 5 ␮M dGTP/dTTP/dATP, 5 units of Klenow fragment, and 16.7 pmol of [␣-32P]dCTP, and the mixture was incubated at 18 °C overnight. To produce strand-specific probes for exon 3 of the APRT gene, a PCR-amplified fragment of the exon 3 region was used as the template and either T or NT strandspecific primer was used to perform separate linear PCR reactions.

RESULTS

Only One Strand of the APRT Gene Is Transcribed in Wild

DISCUSSION