Transcription-coupled repair deficiency and mutations in human mismatch repair genes.

Deficiencies in mismatch repair have been linked to a common cancer predisposition syndrome in humans, hereditary nonpolyposis colorectal cancer (HNPCC), and a subset of sporadic cancers. Here, several mismatch repair-deficient tumor cell lines and HNPCC-derived lymphoblastoid cell lines were found to be deficient in an additional DNA repair process termed transcription-coupled repair (TCR). The TCR defect was corrected in a mutant cell line whose mismatch repair deficiency had been corrected by chromosome transfer. Thus, the connection between excision repair and mismatch repair previously described in Escherichia coli extends to humans. These results imply that deficiencies in TCR and exposure to carcinogens present in the environment may contribute to the etiology of tumors associated with genetic defects in mismatch repair.

Transcription and DNA damage: a link to a kink.

Living organisms are constantly exposed to a variety of naturally occurring and man-made chemical and physical agents that pose threats to health by causing cancer and other illnesses, as well as cell death. One mechanism by which these moieties can exert their toxic effects is by inducing modifications to the genome. Such changes in DNA often result in the formation of nucleotides not normally found in the double helix, bases containing covalent chemical alterations, single- and double-strand breaks, and interstrand and intrastrand cross-links. When these lesions are present during replication, mutations often result in the newly synthesized DNA. Likewise, when such damage occurs in a gene, transcription elongation, and hence expression, can be adversely affected because of pausing or arresting of the RNA polymerase at or near the altered site; this could result in the synthesis of a defective RNA molecule. It has become increasingly clear that transcription and DNA damage are intimately linked, since the removal of certain adducts from the genome is highly dependent on their location. When such lesions are present on the transcribed strand of actively expressed genetic loci, they are better cleared from that strand when compared to the complementary DNA or other quiescent regions. This process is called transcription-coupled DNA repair, and it modulates the mutagenic spectrum of many DNA-damaging agents. Furthermore, based upon evidence from systems in which it is absent, this process has a profound effect on ameliorating the adverse consequences of exposure to many environmentally relevant genotoxins. The precise cellular pathway that mediates the preferential clearance of DNA damage from active genetic loci has not yet been established, but it appears to be effected by a repertoire of proteins that are also involved in other DNA repair pathways and transcription as well as some factors that might be unique to it. Because a cellular process as indispensable as gene expression can be thwarted by the presence of DNA damage, an understanding of the mechanism underlying transcription-coupled DNA repair is relevant to the continued discernment of how environmental genotoxins endanger human health.

Spontaneous and ionizing radiation induced mutations involve large events when selecting for loss of an autosomal locus.

The mouse P19H22 embryonal carcinoma cell line contains two distinct chromosome 8 homologs, one derived from Mus musculus domesticus (M. domesticus) and the other derived from Mus musculus musculus (M. musculus). It also contains a deletion for the M. musculus aprt allele, which is located on chromosome 8. In this study, cells with spontaneous or induced aprt deficiencies were isolated from P19H22 and examined to determine the nature of the mutational events that had occurred. Ultraviolet radiation (UV), ethyl methanesulfonate (EMS), and two forms of ionizing radiation, 137Cs and 252Cf, were used for mutation induction. DNA preparations from the aprt deficient cells were initially screened with a Southern blot analysis and separated into two broad classes: those that had lost the M. domesticus aprt allele and those that had retained it. The overwhelming majority (> 95%) of the spontaneous and ionizing radiation-induced mutants exhibited aprt gene loss, indicating that relatively large events had occurred and that homozygosity for the deleted region was not a lethal event. Loss of heterozygosity for syntenic markers was found to be a common event in cells exhibiting aprt gene loss. In contrast, a majority of the UV-induced mutants (61%) and a substantial minority of the EMS-induced mutants (38%) retained the aprt gene. A sequence analysis confirmed that base-pair substitutions were responsible for this class of mutation. Gene inactivation associated with hypermethylation of the promoter region was found to be a rare event and was not induced by any of the mutagenic agents tested. The results demonstrate the suitability of the P19H22 cell line for mutational studies, particularly those that are large in nature.

Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand.

Nucleotide excision repair helps to ameliorate the lethal and mutagenic consequences of DNA damage by removing helix-distorting lesions from cellular genomes. We have previously analysed the removal of ultraviolet-induced cyclobutane pyrimidine dimers from specific DNA sequences in mammalian cells and demonstrated that transcriptionally active genes are preferentially repaired. Additionally, we found that in rodent and human cells only the transcribed strand of the dihydrofolate reductase gene is selectively repaired. Transcription is blocked by pyrimidine dimers in template DNA and the selective removal of these lesions seems to be important for cell survival after irradiation with ultraviolet light. To determine whether this feature of repair is common to prokaryotes and eukaryotes and better to understand its mechanism, we have investigated repair in the two separate DNA strands of the lactose operon of ultraviolet-irradiated Escherichia coli. We find a dramatic difference in the repair of the two strands only when transcription is induced. Most dimers are removed from the transcribed strand of the induced operon within five minutes of irradiation. In the nontranscribed strand, repair is significantly slower and resembles that found in both strands of the uninduced operon. Thus there seems to be a mechanism that couples nucleotide excision repair and transcription.

Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli.

To improve our understanding of the mechanism that couples nucleotide-excision repair to transcription in expressed genes, we have examined the effects of mutations in several different DNA repair genes on the removal of cyclobutane pyrimidine dimers from the individual strands of the induced lactose operon in UV-irradiated Escherichia coli. As expected, we found little repair in either strand of the lactose operon in strains with mutations in established nucleotide excision-repair genes (uvrA, uvrB, uvrC, or uvrD). In contrast, we found that mutations in either of two genes required for DNA-mismatch correction (mutS and mutL) selectively abolish rapid repair in the transcribed strand and render the cells moderately sensitive to UV irradiation. Similar results were found in a strain with a mutation in the mfd gene, the product of which has been previously shown to be required for transcription-coupled repair in vitro. Our results demonstrate an association between mismatch-correction and nucleotide-excision repair and implicate components of DNA-mismatch repair in transcription-coupled repair. In addition, they may have important consequences for human disease and may enhance our understanding of the etiology of certain cancers which have been associated with defects in mismatch correction.

Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene.

We find a dramatic difference in the efficiency of removal of UV-induced pyrimidine dimers from the transcribed and nontranscribed strands of the dihydrofolate reductase (DHFR) gene in cultured hamster and human cells. In hamster cells, 80% of the dimers are removed from the transcribed strand in 4 hr, but little repair occurs in the nontranscribed strand even after 24 hr. In human cells, repair is significantly faster in the transcribed strand than in the other strand. Furthermore, in the 5' flanking region of the human DHFR gene, selective rapid repair occurs in the opposite DNA strand relative to the transcribed strand of the DHFR gene. This strand is thought to serve as a template for transcription of a divergent transcript. These results have important implications for excision repair pathways and mutagenesis in mammalian cells.

Is high mobility group protein 17 phosphorylated in vivo? Re-examination of the HeLa cell cycle data.

When in vivo [32P] phosphate labeled HMG proteins from unsynchronized HeLa cells are separated by electrophoresis in acid-urea polyacrylamide gels, as opposed to separation in SDS-polyacrylamide, HMG 17 does not show any 32P incorporation. Likewise, no 32P radioactivity was found in HMG 17 protein isolated at different stages of the cell cycle from synchronized cells. By contrast, HMG 14 reveals a previously reported (Bhorjee, J.S. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6944-6948) cell cycle stage-specific dependent phosphorylation with maximum 32P radioactivity in the G2 phase relative to G1. Furthermore, HMG 14 is resolved into multiple electrophoretic forms as phosphoprotein in the acid-urea system. The results presented seriously question the data on the in vivo phosphorylation of HMG 17, and suggest that these be reevaluated.

Preferential DNA repair of an active gene in human cells.

Removal of pyrimidine dimers was measured in defined sequences in human cells amplified for the dihydrofolate reductase (DHFR) gene. We quantitated repair in specific restriction fragments by using the dimer-specific bacteriophage T4 endonuclease V and analysis by Southern blotting. Within 4 hr after 5- or 10-J/m2 UV irradiation, more than 60% of the dimers had been removed from a 20-kilobase fragment that lies entirely within the transcription unit of the DHFR gene and from a 25-kilobase fragment located in the 5' flanking region of the gene. Repair in the overall genome was measured by analyzing cellular DNA treated with T4 endonuclease V in alkaline sucrose gradients. Sixty-nine percent of the dimers were removed from the genome overall within 24 hr after irradiation, but only 25% were removed within 4 hr and 38% were removed within 8 hr. These results demonstrate a strong preferential rate of removal of dimers from the 50-kilobase region that includes the transcriptionally active DHFR gene compared to that in total cellular DNA. We confirmed that DHFR-containing DNA is repaired more rapidly than bulk DNA by using an approach that provides a direct comparison between repair in specific sequences and repair in total cellular DNA. We also show that the DHFR-containing sequences are repaired more rapidly than the nontranscribed repetitive alpha DNA sequences. Our finding of preferential early repair in a transcriptionally active region in overall repair-proficient cells suggests that selective dimer removal from active sequences may be a general characteristic of mammalian DNA repair.