Rodney S. Nairn

THE BIOLOGY OF THE (6–4) PHOTOPRODUCT

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.

Involvement of Nucleotide Excision Repair in a Recombination-Independent and Error-Prone Pathway of DNA Interstrand Cross-Link Repair

ABSTRACT 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 recombination-independent 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.

Nucleotide Excision Repair- and Polymerase η-Mediated Error-Prone Removal of Mitomycin C Interstrand Cross-Links

ABSTRACT 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, recombination-independent 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 η 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.

Role of ERCC1 in removal of long non‐homologous tails during targeted homologous recombination

The XpF/Ercc1 structure‐specific endonuclease performs the 5′ incision in nucleotide excision repair and is the apparent mammalian counterpart of the Rad1/Rad10 endonuclease from Saccharomyces cerevisiae. In yeast, Rad1/Rad10 endonuclease also functions in mitotic recombination. To determine whether XpF/Ercc1 endonuclease has a similar role in mitotic recombination, we targeted the APRT locus in Chinese hamster ovary ERCC1+ and ERCC1− cell lines with insertion vectors having long or short terminal non‐homologies flanking each side of a double‐strand break. No substantial differences were evident in overall recombination frequencies, in contrast to results from targeting experiments in yeast. However, profound differences were observed in types of APRT+ recombinants recovered from ERCC1− cells using targeting vectors with long terminal non‐homologies—almost complete ablation of gap repair and single‐reciprocal exchange events, and generation of a new class of aberrant insertion/deletion recombinants absent in ERCC1+ cells. These results represent the first demonstration of a requirement for ERCC1 in targeted homologous recombination in mammalian cells, specifically in removal of long non‐homologous tails from invading homologous strands.

Ultraviolet A does not induce melanomas in a Xiphophorus hybrid fish model

We examined the wavelength dependence of ultraviolet (UV) ra-diation (UVR)-induced melanoma in a Xiphophorus backcross hybrid model previously reported to be susceptible to melanoma induction by ultraviolet A (UVA) and visible light. Whereas ultraviolet B (UVB) irradiation of neonates yielded high frequencies of melanomas in pigmented fish, UVA irradiation resulted in melanoma frequencies that were not significantly different from unirradiated fish. Spontaneous and UV-induced melanoma frequencies correlated with the degree of pigmentation as expected from previous studies, and the histopathology phenotypes of the melanomas were not found in significantly different proportions in UV-treated and -untreated tumor-bearing fish. Our results support the conclusion that a brief early-life exposure to UVB radiation causes melanoma formation in this animal model. These data are consistent with an essential role for direct DNA damage, including cyclobutane dimers and (6-4) photoproducts, in the etiology of melanoma.

Localization of a CDKN2 gene in linkage group V of Xiphophorus fishes defines it as a candidate for the DIFF tumor suppressor

The Xiphophorus hybrid melanoma model represents one of the earliest reported cases of genetically regulated tumor susceptibility. Melanoma formation in Xiphophorus hybrids may be explained by the inheritance of two genes: a sex‐linked oncogene, Xmrk, and a putative tumor suppressor locus, termed DIFF, located in Linkage Group V (LG V). Several genetic mapping procedures were used to produce a new Xiphophorus LG V map with 20 loci. All markers, particularly a recently cloned Xiphophorus CDKN2 gene family member, called CDKN2X, were tested for associations of genotype with degree of macromelanophore pigment pattern modification and susceptibility to melanoma formation in backcross hybrids of seven genetic types, involving 1,110 fish and three pigment patterns. Highly significant associations of CDKN2X genotypes with such phenotypic effects suggests that this gene is a strong candidate for the classically defined DIFF tumor suppressor gene. Because published results have documented the involvement of the CDKN2A (p16, MTS1, and INK4A) tumor suppressor gene in human melanoma formation, the possibility of CDKN2 genes acting as tumor suppressors in both man and Xiphophorus is likely. Genes Chromosomes Cancer 22:210–220, 1998.

NONMAMMALIAN MODELS FOR SUNLIGHT CARCINOGENESIS : GENETIC ANALYSIS OF MELANOMA FORMATION IN XIPHOPHORUS HYBRID FISH

Abstract— Genetic hybrids of Xiphophorus fishes have been used for decades to study heritable melanoma formation. In these models, overexpression of pigmentation patterns from melanin‐producing pigment cells can lead to genetically regulated melanoma formation in backcross hybrids. In the best studied of these models, the Gordon‐Kosswig hybrid melanoma, tumors form spontaneously in all individuals of a subset of backcross hybrids between the platyflsh Xiphophorus maculatus Jp 163 A and the swordtail species Xiphophorus helleri. Backcross hybrids susceptible to melanoma formation inherit a sex‐linked oncogene, Xmrk, associated with the spotted dorsal (Sd) pigment pattern and have lost both copies of an autosomal gene, DIFF, from the X. maculatus parent. Spontaneous melanoma formation conforms to simple, two‐gene Mendelian inheritance in which DIFF behaves as a recessive tumor suppressor gene. Recently, Xiphophorus hybrids in which melanomas can be induced by UV and near‐UV visible light exposure have been described. We report here results of genetic linkage analysis of one of these Xiphophorus light‐inducible hybrid melanoma models, in backcross hybrids between the two platyflsh species X. maculatus Jp 163 B and Xiphophorus couchianus. Our linkage results provide the first estimate of recombination between the tumor suppressor locus, DIFF, and glycerate‐2‐dehydrogenase (GLYDH) in Xiphophorus linkage group V. Also, they demonstrate that DIFF regulates hyperplasia of spotted side (Sp) pigment cells in this hybrid model, analogous to its regulation of hyperplasia of Sd pigment cells in the “classical” Gordon‐Kosswig hybrid. Joint segregation analyses of melanoma‐bearing fish indicate that segregation of DIFF is genetically linked to melanoma induction by 405 nm light in this model but that induction of melanomas by UV wavelengths apparently does not depend on segregation of the DIFF locus.

Genetic and environmental melanoma models in fish

Experimental animal models are extremely valuable for the study of human diseases, especially those with underlying genetic components. The exploitation of various animal models, from fruitflies to mice, has led to major advances in our understanding of the etiologies of many diseases, including cancer. Cutaneous malignant melanoma is a form of cancer for which both environmental insult (i.e., UV) and hereditary predisposition are major causative factors. Fish melanoma models have been used in studies of both spontaneous and induced melanoma formation. Genetic hybrids between platyfish and swordtails, different species of the genus Xiphophorus, have been studied since the 1920s to identify genetic determinants of pigmentation and melanoma formation. Recently, transgenesis has been used to develop zebrafish and medaka models for melanoma research. This review will provide a historical perspective on the use of fish models in melanoma research, and an updated summary of current and prospective studies using these unique experimental systems.

Loss of thymine dimers from mammalian cell DNA. The kinetics for antibody-binding sites are not the same as that for T4 endonuclease V sites

Antiserum specific for thymine-containing dimers was used to assay DNA isolated from ultraviolet-irradiated cells following different repair periods. A 50% loss in antibody-binding sites was evident 1 h post-irradiation, and within 4 h 80% of the sites were removed. This result contrasts with data obtained with dimer-specific T4 endonuclease V and does not appear to be due to masking of the dimers by repair enzymes. T4 endonuclease V treatment of ultraviolet-irradiated DNA at 0 degree C resulted in conversion of the thymine dimers to apyrimidinic sites. This did not result in loss of antigenicity in either PM2 or CHO cell DNA. Likewise, treatment of ultraviolet-irradiated CHO cell DNA with T4 endonuclease at 37 degrees C did not change its antigenicity. These results suggest that aglycosylation of the dimers is not responsible for their inability to bind dimer-specific antibody 2-4 h post-irradiation. The possibility that T4 endonuclease V and the antiserum have different specificities for different dimers is discussed.

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.