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...

We have determined the spectrum of base-pair substitution mutations induced in the lad gene of a uvrB-strain of Escherichia coli by two polycyclic aromatic hydrocarbons-(±)7a,8fi-dihydroxy-9p, 10I3-epoxy-7,8,9,10 tetrahydrobenzo-[a]pyrene (BPDE), and 3,4-epoxycydopenta [cd]pyrene (CPPE). Approximately 10% of all lad mutations induced by either BPDE or CPPE are nonsense mutations, suggesting that base-pair substitutions are a large fraction of the mutational events induced by these agents in the uvrB-bacteria. Both carcinogens specifically induced the G-C -* TA and, to a lesser extent, the A-T -T-A transversions. One possible mechanism for transversion induction at G-C sites by BPDE might involve carcinogen binding to the exocyclic amino group of guanine in the template strand followed by a rotation of the modified base around its glycosylic bond from the anti to the syn conformation. This could allow specific pairing of modified bases with an imino tautomer of adenine.Although the mutagenicity of most carcinogens is well documented (1), little is known about the spectrum of mutagenic events that carcinogens are capable of inducing. The most widely utilized mutagenicity test, the Ames test (1), detects the ability of chemicals to revert any one of three different point mutations (two frameshift mutations and a base-substitution mutation) but does not yet provide information about the specific changes in DNA sequence that are induced at these loci. Because there is a wide range of possible base sequence alterations it seems of fundamental importance to document the spectrum of mutations induced in vivo by carcinogens. Miller and his colleagues have developed a genetic system in Escherichia coli which is useful for rapidly and rigorously identifying base-pair substitutions that generate nonsense mutations at 64 sites within the lad gene (2). This system detects all possible base-pair substitution events with the exception of the A-T -> G-C transition. We have begun to use the lad system to explore the spectrum of base-pair substitutions induced in vivo by carcinogens.In the study reported here we focused on two polycyclic aromatic hydrocarbons which are environmental carcinogens: benzo[a]pyrene (BP) and cyclopenta[cd]pyrene (CPP) (3-5).Many carcinogens are metabolized to highly reactive species which bind covalently to DNA (6) and induce mutations in bacteria or in mammalian cells (7,8). The reactive metabolite of BP that accounts for most of the binding to DNA is a trans diol epoxide, (±)7a,8,3dihydroxy-9,, 10,3epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDE) (9). picted in Fig. 1 (10). Adducts of the trans diol epoxide with the exocyclic N6 amino group of adenine (11) and with cytosine (12) have also been reported. There is also indirect evidence that an adduct may be formed between the trans diol epoxide and the N7 position of guanine (13). CPP adducts to DNA have not been identified. Which of the BPDE-DNA adducts represent biologically significant premutational lesions? The mutational spectrum induced b...

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Examining global effects of toxic metals on gene expression can be useful for elucidating patterns of biological response, discovering underlying mechanisms of toxicity, and identifying candidate metal-specific genetic markers of exposure and response. Using a 1,200 gene nylon array, we examined changes in gene expression following low-dose, acute exposures of cadmium, chromium, arsenic, nickel, or mitomycin C (MMC) in BEAS-2B human bronchial epithelial cells. Total RNA was isolated from cells exposed to 3 M Cd(II) (as cadmium chloride), 10 M Cr(VI) (as sodium dichromate), 3 g/cm2 Ni(II) (as nickel subsulfide), 5 M or 50 M As(III) (as sodium arsenite), or 1 M MMC for 4 hr. Expression changes were verified at the protein level for several genes. Only a small subset of genes was differentially expressed in response to each agent: Cd, Cr, Ni, As (5 M), As (50 M), and MMC each differentially altered the expression of 25, 44, 31, 110, 65, and 16 individual genes, respectively. Few genes were commonly expressed among the various treatments. Only one gene was altered in response to all four metals (hsp90), and no gene overlapped among all five treatments. We also compared low-dose (5 M, noncytotoxic) and high-dose (50 M, cytotoxic) arsenic treatments, which surprisingly, affected expression of almost completely nonoverlapping subsets of genes, suggesting a threshold switch from a survival-based biological response at low doses to a death response at high doses.