The (6-4) photoproduct is an important determinant of the lethal and mutagenic effects of UV irradiation of biological systems. The removal of this lesion appears to correlate closely with the early DNA repair responses of mammalian cells, including DNA incision events, repair synthesis and removal of replication blocks. The processing of (6-4) photoproducts and cyclobutane dimers appears to be enzymatically coupled in bacteria and most mammalian cell lines examined (i.e. a mutation affecting the repair of one lesion also often affects the other), although exceptions exist in which repair capacity may be evident for one photoproduct and not the other (e.g. UV61 and the XP revertant cell line). These differences in the processing of the two photoproducts in some cell lines of human and rodent origin suggest that in mammalian cells, different pathways for the repair of (6-4) photoproducts and cyclobutane dimers may be used. This observation is further supported by pleiotropic repair phenotypes such as those observed in CHO complementation class 2 mutants (e.g., UV5, UVL-1, UVL-13, and V-H1). Indirect data, from HCR of UV irradiated reported genes and the cytotoxic responses of UV61, suggest that the (6-4) photoproduct is cytotoxic in mammalian cells and may account for 20 to 30% of the cell killing after UV irradiation of rodent cells. Cytotoxicity of the (6-4) photoproduct may be important in the etiology of sunlight-induced carcinogenesis, affecting mutagenesis as well as tumorigenesis. The intricate photochemistry of the (6-4) photoproduct, its formation and photoisomerization, is in itself extremely interesting and may also be relevant to sunlight carcinogenesis. The data reviewed in this article support the notion that the (6-4) photoproduct and its Dewar photoisomer are important cytotoxic determinants of UV light. The idea that the (6-4) photoproduct is an important component in the spectrum of UV-induced cytotoxic damage may help clarify our understanding of why rodent cells survive the effects of UV irradiation as well as human cells, without apparent cyclobutane dimer repair in the bulk of their DNA. The preferential repair of cyclobutane dimers in essential genes has been proposed to account for this observation (Bohr et al., 1985, 1986; Mellon et al., 1986). The data reviewed here suggest that understanding the repair of a prominent type of noncyclobutane dimer damage, the (6-4) photoproduct, may also be important in resolving this paradox.

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...

DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombinationindependent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.Alkylating agents were among the first compounds found to be efficacious in cancer therapy and remain important components of many modern chemotherapeutic regimens (38). Many members of this class of drugs have bifunctional groups that can react with both strands of the DNA helix and thus form interstrand and intrastrand cross-links. As profound blocks for both transcription and DNA replication, interstrand cross-links (ICLs) appear to represent the primary cytotoxic lesion induced by most bifunctional alkylating agents.Repair of DNA ICLs has been studied extensively in Escherichia coli (11,12). Both genetic and biochemical evidence has established a combined nucleotide excision repair (NER)-recombination mechanism for the error-free repair of ICLs, in which the gap, created by the Uvr(A)BC excinuclease, is repaired through recA-mediated recombination with a lesionfree homologous chromosome as the donor (10, 37, 41). Although the NER-recombination pathway appears to be the primary mechanism of cross-link repair in E. coli, recent evidence has suggested a recombination-independent pathway for cross-link repair in which the gap created by the uvr(A)BC excinuclease is repaired by translesion bypass in order to circumvent a d...

Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RAD10 gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1 ؊ and ERCC1 ؉ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT ؉ tandem duplication could give rise to APRT ؊ cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT ؊ products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1 ؊ cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1 ؊ cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1 ؊ cells are repaired by illegitimate recombination.