Interstrand cross-links (ICLs) make up a unique class of DNA lesions in which both strands of the double helix are covalently joined, precluding strand opening during replication and transcription. The repair of DNA ICLs has become a focus of study since ICLs are recognized as the main cytotoxic lesion inflicted by an array of alkylating compounds used in cancer treatment. As is the case for double-strand breaks, a damage-free homologous copy is essential for the removal of ICLs in an error-free manner. However, recombinationindependent mechanisms may exist to remove ICLs in an error-prone fashion. We have developed an in vivo reactivation assay that can be used to examine the removal of site-specific mitomycin C-mediated ICLs in mammalian cells. We found that the removal of the ICL from the reporter substrate could take place in the absence of undamaged homologous sequences in repair-proficient cells, suggesting a cross-link repair mechanism that is independent of homologous recombination. Systematic analysis of nucleotide excision repair mutants demonstrated the involvement of transcription-coupled nucleotide excision repair and a partial requirement for the lesion bypass DNA polymerase encoded by the human POLH gene. From these observations, we propose the existence of a recombination-independent and mutagenic repair pathway for the removal of ICLs in mammalian cells.A DNA interstrand cross-link (ICL) is formed when both strands of the double helix are covalently joined by a single molecule. Since ICLs effectively prevent strand separation, essential metabolic functions of DNA such as transcription, replication, and recombination are severely blocked by these lesions. The formation of DNA ICLs appears to be an essential prerequisite for the potent cytotoxicity and antitumor activity of a large array of chemotherapeutic compounds used in cancer treatment (41).In Escherichia coli and lower eukaryotes, the repair of ICLs is carried out primarily by a combination of the nucleotide excision repair (NER) and homologous recombination pathways. In a model proposed by Cole et al. (9, 10) based on genetic evidence, the NER mechanism introduces incisions flanking the site of the cross-link on the same strand. The resulting gap is then repaired by using a lesion-free homologous chromosome as a donor via the recA-dependent homologous recombination pathway. Subsequent biochemical analyses fully confirmed that the removal of ICLs in E. coli is mediated by both NER and homologous recombination (39,44,45). Similarly, with Saccharomyces cerevisiae, a group of RAD3 mutants (deficient in NER) and a group of RAD52 mutants (deficient in homologous recombination) are hypersensitive to the killing of bifunctional alkylating agents, suggesting that both pathways are essential for the repair of ICLs (21,28,30,38). These observations also indicated the presence of a combination of NER and homologous-recombination mechanisms in ICL repair. More recently, direct evidence of psoralen ICL-induced homologous recombination in budding yeast h...

Arsenic (As) contamination of drinking water is considered a serious worldwide environmental health threat that is associated with increased disease risks including skin, lung, bladder, and other cancers; type 2 diabetes; vascular and cardiovascular diseases; reproductive and developmental effects; and neurological and cognitive effects. Increased health risks may occur at as low as 10-50 ppb, while biological effects have been observed in experimental animal and cell culture systems at much lower levels. We previously reported that As is a potent endocrine disruptor, altering gene regulation by the closely related glucocorticoid, mineralocorticoid, progesterone, and androgen steroid receptors (SRs) at concentrations as low as 0.01 microM ( approximately 0.7 ppb). Very low doses enhanced hormone-mediated gene transcription, whereas slightly higher but still noncytotoxic doses were suppressive. We report here that As also disrupts the more distally related estrogen receptor (ER) both in vivo and in cell culture. At noncytotoxic doses (1-50 micromol/kg arsenite) As strongly suppressed ER-dependent gene transcription of the 17beta-estradiol (E2)-inducible vitellogenin II gene in chick embryo liver in vivo. In cell culture, noncytotoxic levels (0.25-3 microM, approximately 20-225 ppb) of As significantly inhibited E2-mediated gene activation of an ER-regulated reporter gene and the native ER-regulated GREB1 gene in human breast cancer MCF-7 cells. While the effects of As on ER-dependent gene regulation were generally similar to As effects on the other SRs, there were specific differences, particularly the lack of significant enhancement at the lowest doses, that may provide insights into possible mechanisms.

Chronic human exposure to nonovertly toxic doses of arsenic is associated with an increased risk of cancer. Although its carcinogenic mechanism is still unknown, arsenic does not directly cause DNA damage or mutations and is therefore thought to act principally as a co-mutagen, co-carcinogen, and/or tumor promoter. Previous studies in our laboratory demonstrated that effects of low-dose arsenic (III) (arsenite) on expression of the hormone-regulated phosphoenolpyruvate carboxykinase (PEPCK) gene were strongly associated with the glucocorticoid receptor (GR)-mediated regulatory pathway. We therefore examined specifically the effects of arsenite on the biochemical function of GR in hormone-responsive H4IIE rat hepatoma cells. Completely noncytotoxic arsenite treatments (0.3-3.3 microM) significantly decreased dexamethasone-induced expression of transiently transfected luciferase constructs containing either an intact hormone-responsive promoter from the mammalian PEPCK gene or two tandem glucocorticoid response elements (GRE). Western blotting and confocal microscopy of a green fluorescent protein-tagged-GR fusion protein demonstrated that arsenite pretreatment did not block the normal dexamethasone-induced nuclear translocation of GR. These data indicate that nontoxic doses of arsenite can interact directly with GR complexes and selectively inhibit GR-mediated transcription, which is associated with altered nuclear function rather than a decrease in hormone-induced GR activation or nuclear translocation.