The homologous recombination protein RAD51D protects the genome from large deletions.

Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that protects the genome from chromosomal instability. RAD51 mediator proteins (i.e. paralogs) are critical for efficient HR in mammalian cells. However, how HR-deficient cells process DSBs is not clear. Here, we utilized a loss-of-function HR-reporter substrate to simultaneously monitor HR-mediated gene conversion and non-conservative mutation events. The assay is designed around a heteroallelic duplication of the Aprt gene at its endogenous locus in isogenic Chinese hamster ovary cell lines. We found that RAD51D-deficient cells had a reduced capacity for HR-mediated gene conversion both spontaneously and in response to I-SceI-induced DSBs. Further, RAD51D-deficiency shifted DSB repair toward highly deleterious single-strand annealing (SSA) and end-joining processes that led to the loss of large chromosomal segments surrounding site-specific DSBs at an exceptionally high frequency. Deletions in the proximity of the break were due to a non-homologous end-joining pathway, while larger deletions were processed via a SSA pathway. Overall, our data revealed that, in addition to leading to chromosomal abnormalities, RAD51D-deficiency resulted in a high frequency of deletions advancing our understanding of how a RAD51 paralog is involved in maintaining genomic stability and how its deficiency may predispose cells to tumorigenesis.

Use of gene targeting to study recombination in mammalian cell DNA repair mutants.

The study of gene function has been greatly facilitated by the development of strategies to modify genomic DNA. Gene targeting is one of the most successfully applied techniques used to examine the roles of specific genes in a wide variety of model systems from yeast to mammals. Our laboratory has pioneered the use of the Chinese hamster ovary (CHO) cell culture model system to study pathways of DNA repair and recombination at the hemizygous CHO APRT locus. By using a simple and effective gene targeting method, we have generated a number of DNA repair-deficient cell lines that have been used in targeted recombination experiments to investigate pathways of recombinational repair in somatic mammalian cells. These methods can be readily customized to generate a variety of cell lines deficient in specific genes of interest and can be applied to study the roles of other DNA repair proteins in pathways of mammalian recombinational repair.

Effects of varying gene targeting parameters on processing of recombination intermediates by ERCC1-XPF.

The ERCC1-XPF structure-specific endonuclease is necessary for correct processing of homologous recombination intermediates requiring the removal of end-blocking nonhomologies. We previously showed that targeting the endogenous CHO APRT locus with plasmids designed to generate such intermediates revealed defective recombination phenotypes in ERCC1 deficient cells, including suppression of targeted insertion and vector correction recombinants and the generation of a novel class of aberrant recombinants through a deletogenic mechanism. In the present study, we examined some of the mechanistic features of ERCC1-XPF in processing recombination intermediates by varying gene targeting parameters. These included altering the distance between the double-strand break (DSB) in the targeting vector and the inactivating mutation in the APRT target gene, and changing the position of the target gene mutation relative to the DSB to result in target mutations that were either upstream or downstream from the DSB. Increasing the distance from the DSB in the targeting vector to the chromosomal target gene mutation resulted in an ERCC1 dependent decrease in the efficiency of gene targeting from intermediates presenting lengthy end-blocking nonhomologies. This decrease was accompanied by a shift in the distribution of recombinant classes away from target gene conversions to targeted insertions in both wild-type and ERCC1 deficient cells, and a dramatic increase in the proportion of aberrant recombinants in ERCC1 deficient cells. Changing the position of the target gene mutation relative to the DSB in the plasmid also altered the distribution of targeted insertion subclasses recovered in wild-type cells, consistent with two-ended strand invasion followed by resolution into crossover-type products and vector integration. Our results confirm expectations from studies of Rad10-Rad1 in budding yeast that ERCC1-XPF activity affects conversion tract length, and provide evidence for the mechanism of generation of the novel, aberrant recombinant class first described in our previous study.

Depletion of Werner helicase results in mitotic hyperrecombination and pleiotropic homologous and nonhomologous recombination phenotypes.

Werner syndrome (WS) is a rare, segmental progeroid syndrome caused by defects in the WRN gene, which encodes a RecQ helicase. WRN has roles in many aspects of DNA metabolism including DNA repair and recombination. In this study, we exploited two different recombination assays previously used to describe a role for the structure-specific endonuclease ERCC1-XPF in mitotic and targeted homologous recombination. We constructed Chinese hamster ovary (CHO) cell lines isogenic with the cell lines used in these previous studies by depleting WRN using shRNA vectors. When intrachromosomal, mitotic recombination was assayed in WRN-depleted CHO cells, a hyperrecombination phenotype was observed, and a small number of aberrant recombinants were generated. Targeted homologous recombination was also examined in WRN-depleted CHO cells using a plasmid-chromosome targeting assay. In these experiments, loss of WRN resulted in a significant decrease in nonhomologous integration events and ablation of recombinants that required random integration of the corrected targeting vector. Aberrant recombinants were also recovered, but only from WRN-depleted cells. The pleiotropic recombination phenotypes conferred by WRN depletion, reflected in distinct homologous and nonhomologous recombination pathways, suggest a role for WRN in processing specific types of homologous recombination intermediates as well as an important function in nonhomologous recombination.

Multiple roles of ERCC1-XPF in mammalian interstrand crosslink repair.

DNA interstrand crosslinks (ICLs) are among the most deleterious cytotoxic lesions encountered by cells, mainly due to the covalent linkage these lesions create between the two strands of DNA which effectively blocks replication and transcription. Although ICL repair in mammalian cells is not fully understood, processing of these lesions is thought to begin by "unhooking" at the site of the damaged base accompanied by the generation of a double strand break and ultimately repair through translesion synthesis and homologous recombination. A key player in this repair process is the heterodimeric protein complex ERCC1-XPF. Although some models of ICL repair restrict ERCC1-XPF activity to the unhooking step, recent data suggest that this protein complex acts in additional downstream steps. Here, we review the evidence implicating ERCC1-XPF in multiple steps of ICL repair.

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.

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.

Xmrk in medaka: a new genetic melanoma model.

Melanoma is the most lethal form of skin cancer, and its incidence is rising rapidly (Chin et al., 2006; Linos et al., 2009). Highly aggressive, metastatic melanoma is notoriously resistant to chemotherapy, and early detection is critical for surgical excision (Gray-Schopfer et al., 2007). A detailed knowledge of the cellular, molecular, and genetic events during melanoma progression is highly relevant to both diagnosis and the development of new therapies. Animal models, such as the one described in this issue by Schartl and colleagues, are important tools for identifying the genetic and environmental factors that contribute to melanoma development, as well as identifying and testing new therapeutic strategies.

Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells.

Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells.

Etiology of MNU-induced melanomas in Xiphophorus hybrids.

Genetic hybrids of the genus Xiphophorus have historically been useful models for study of the genetic aspects of tumor formation. In the most studied Xiphophorus tumor model, two-gene loci, XMRK and DIFF, are implicated as critical both to UV-induced and spontaneous melanoma formation in BC(1) hybrids of crosses between X. maculatus and X. helleri, with X. helleri as the recurrent backcross parent. In addition to UV, the direct-acting carcinogen N-methyl-N-nitrosourea (MNU) has been used to induce tumors in Xiphophorus BC(1) hybrids from several cross types. In the present study, we address the hypothesis that excess melanomas in MNU-treated BC(1) hybrids may have been generated by direct mutation of CDKN2AB, a candidate gene for DIFF. MNU treatment of F(1) and BC(1) hybrid fish significantly increased tumor incidence at 6 months; however, no association was found between MNU-induced tumor formation and zygosity of the candidate tumor tumor-suppressor CDKN2AB in BC(1) hybrids, consistent with previously reported results. Sequence analysis of the X. maculatus CDKN2AB locus of heterozygous individuals (both BC(1) and F(1) hybrids) did not reveal any mutations caused by MNU, suggesting that the mechanism of MNU-induced melanoma formation in this Xiphophorus model does not involve direct mutation of CDKN2AB but may result from mutation of other critical genes.

Processing of triplex-directed psoralen DNA interstrand crosslinks by recombination mechanisms.

Gene targeting via homologous recombination (HR) is an important application in biotechnology and medicine. However, in mammalian cells HR is much less efficient than random integration. Triplex-forming oligonucleotides (TFOs) linked to DNA damaging agents (e.g. psoralen) can stimulate HR, providing the potential to improve gene therapy applications. To elucidate factors affecting TFO-directed psoralen interstrand crosslink (ICL)-induced recombination, we constructed a series of plasmids with duplicated supF reporter genes, each containing an inactivating deletion, to measure HR frequencies in mammalian cells. Our results indicated that TFO-directed ICL-induced recombination frequencies were higher in the plasmids with larger distances between duplicated supF genes than with a smaller separation distance. However, the position of the ICL relative to the reporter genes did not affect HR frequencies. Recombination spectra were altered by the distance between supF copies. Although single-strand annealing (SSA) recombinants were predominant in all plasmid substrates, the plasmid with the shortest interval (60 bp) revealed a significant proportion of gene conversions (GCs). GCs occurred exclusively in the gene containing the shortest deletion, regardless of the distance between supF genes, ICL position or deletion orientation. Our analyses indicated that SSA is the predominant mechanism of ICL processing of these substrates in mammalian cells.

Characterization of CHO XPF mutant UV41: influence of XPF heterozygosity on double-strand break-induced intrachromosomal recombination.

The UV hypersensitive CHO cell mutant UV41 is the archetypal XPF mammalian cell mutant, and was essential for cloning the human nucleotide excision repair (NER) gene XPF by DNA transfection and rescue. The ERCC1 and XPF genes encode proteins that form the heterodimer responsible for making incisions required in NER and the processing of certain types of recombination intermediates. In this study, we cloned and sequenced the CHO cell XPF cDNA, determining that the XPF mutation in UV41 is a +1 insertion in exon 8 generating a premature stop codon at amino acid position 499; however, the second allele of XPF is apparently unaltered in UV41, resulting in XPF heterozygosity. XPF expression was found to be several-fold lower in UV41 compared to its parental cell line, AA8. Using approaches we previously developed to study intrachromosomal recombination in CHO cells, we modified UV41 and its parental cell line AA8 to allow site-specific gene targeting at a Flp recombination target (FRT) in intron 3 of the endogenous adenine phosphoribosyltransferase (APRT) locus. Using FLP/FRT targeting, we integrated a plasmid containing an I-SceI endonuclease sequence into this site in the paired cell lines to generate a heteroallelic APRT duplication. Frequencies of intrachromosomal recombination between APRT heteroalleles and the structures of resulting recombinants were analyzed after I-SceI induction of site-specific double-strand breaks (DSBs) in a non-homologous insertion contained within APRT homology. Our results show that I-SceI induced a small proportion of aberrant recombinants reflecting DSB-induced deletions/rearrangements in parental, repair-proficient AA8 cells. However, in XPF mutant UV41, XPF heterozygosity is responsible for a similar, but much more pronounced genomic instability phenotype, manifested independently of DSB induction. In addition, gene conversions were suppressed in UV41 cells compared to wild-type cells. These observations suggest that UV41 exhibits a genomic instability phenotype of aberrant recombinational repair, confirming a critical role for XPF in mammalian cell recombination.

Melanoma susceptibility and cell cycle genes in Xiphophorus hybrids.

Xiphophorus interspecies hybrids provide genetically defined models of both spontaneous and inducible melanomagenesis. In both models, backcrossing F(1) hybrids of different strains of X. maculatus and X. helleri to a X. helleri parental fish results in segregation of melanoma susceptibility, fitting a Mendelian two-gene inheritance model. The sex-linked Xmrk oncogene is required for melanoma development in both crosses. The Xiphophorus CDKN2A/B gene, which is homologous to mammalian CDKN2A/B cyclin-dependent kinase inhibitors (p16 and p15), is a candidate melanoma susceptibility gene. In this model, tumor susceptibility segregates with homozgyosity for CDKN2A/B from the recurrent X. helleri parent in backcross hybrids. We found that both CDKN2A/B mRNA and protein are highly overexpressed in melanoma. Because the p13 protein product of CDKN2A/B is a putative regulator of the G1 checkpoint, we investigated expression of other components of Xiphophorus G1 checkpoint control. By real-time PCR analysis, retinoblastoma gene (RB) is consistently expressed twofold higher in both tumors and melanized skin than in normal tissue, indicating that RB is not downregulated by the overexpression of CDKN2A/B in Xiphophorus melanoma. We also found a significant correlation between the quantitative level of CDKN2A/B and Xmrk RNA in tumors, suggesting a functional relationship between Xmrk and CDKN2A/B expression. Although X. helleri CDKN2A/B protein contains a non-conservative substitution, the biochemical function appears to show little overt defect. These studies indicate that in Xiphophorus melanoma, CDKN2A/B is functionally insufficient to mediate cell-cycle arrest in the presence of Xmrk.

Regulation of CDKN2A/B and Retinoblastoma genes in Xiphophorus melanoma.

Xiphophorus interspecies hybrids provide several well-characterized genetic models of melanoma susceptibility. The Xiphophorus CDKN2A/B gene, homologous to mammalian CDKN2A/B cyclin-dependent kinase inhibitors (p16 and p15), is a candidate tumor susceptibility gene in these models. Using real-time PCR and Western blot analysis, we analyzed expression of CDKN2A/B in spontaneous and UV-induced primary melanomas from individual backcross hybrid fish. We found that CDKN2A/B mRNA is highly expressed in melanomas (18-fold), relative to other fish tissues. Expression is also elevated, to a lesser extent (9.5-fold), in melanized skin from tumor-bearing fish. However, quantitative levels of CDKN2A/B mRNA in tumors varied considerably and positively correlated with expression of the Xmrk oncogene, suggesting possible functional interaction between Xmrk and CDKN2A/B expression. As a homolog corresponding to members of the mammalian CDKN2 family which regulate cell cycle progression at the G1 checkpoint, the CDKN2A/B p13 protein is a putative regulator of the G1 checkpoint apparatus in Xiphophorus. Since CDKN2A is often observed to be inversely regulated compared to RB in some human tumors, and is capable of transcriptionally regulating RB in human ovarian tumors, we cloned the Xiphophorus maculatus RB cDNA and analyzed RB expression by real-time PCR and Western blot analysis in the fish melanomas. These experiments were designed to ascertain whether CDKN2A/B and RB expression were inversely correlated. Our results indicate that RB mRNA was consistently expressed at only a 2-fold higher level in both tumors and melanized skin than in muscle. Qualitatively similar results were obtained for protein expression. These results collectively suggest that (i) Xmrk and CDKN2A/B may be co-regulated at the transcriptional level, and (ii) there is little, if any, alteration of RB expression in Xiphophorus melanomas.

Use of gene targeting to study recombination in mammalian cell DNA repair mutants.

Gene targeting by homologous recombination in mammalian cells is an important tool for generating genetically modified mice used for modeling human diseases. Gene targeting approaches are also useful for studying the mechanisms of homologous recombination. We have developed gene targeting methods that we have specifically used to investigate the mechanisms of recombination in cultured mammalian cells. In this chapter, we describe the generation of Chinese hamster ovary (CHO) cell gene disruption ("knockout") mutants in the repair/recombination gene ERCC1. Using this approach, we have constructed pairs of isogenic ERCC1-proficient and -deficient (null) CHO cell lines and used them as recipients for gene targeting assays in which a hemizygous mutant hamster adenine phosphoribosyltransferase (APRT) locus is corrected by homologous recombination with plasmid vectors containing hamster APRT DNA sequence homologous to the target gene in each cell line. The configuration of the targeting vector leads to experimental outcomes in which certain classes of APRTrecombinants are over- or under-represented depending on the repair gene status of the transfection recipient. We describe methods both for targeted gene knockout of ERCC1, and for APRT targeted gene correction by homologous recombination, and some of our experimental results using these approaches.

Photocarcinogenesis in Xiphophorus hybrid models.

The poeciliid fish genus Xiphophorus provides important models for investigating the etiology and genetics of sunlight-induced melanoma. Interspecific hybrids generated among platyfish and swordtails have been used as genetic tumor models, particularly for cutaneous malignant melanoma (CMM), for more than 6 decades. Oncogene and tumor suppressor gene involvement in a variety of spontaneous and carcinogen-induced tumors has been and continues to be extensively studied. Select hybrids develop melanoma spontaneously or after acute or chronic exposure to ultraviolet radiation. Many scientists believe that the etiology of CMM and particularly its initiation is different from other types of sunlight-induced skin cancers, and may involve free radical chemistry rather than the direct absorption of UVB by DNA. Xiphophorus offers a unique platform to scrutinize this question and determine the types of DNA damage that are involved, the solar wavelength ranges that are important, and the role of DNA repair genes in early tumorigenesis. The diverse photochemical and photobiological responses observed in the different Xiphophorus species and interspecies hybrids suggest that heritable traits governing DNA damage induction and repair may be involved in the susceptibility of Xiphophorus hybrids to melanomagenesis.

Triplex targeted genomic crosslinks enter separable deletion and base substitution pathways.

We have synthesized triple helix forming oligonucleotides (TFOs) that target a psoralen (pso) interstrand crosslink to a specific chromosomal site in mammalian cells. Mutagenesis of the targeted crosslinks results in base substitutions and deletions. Identification of the gene products involved in mutation formation is important for developing practical applications of pso-TFOs, and may be informative about the metabolism of other interstrand crosslinks. We have studied mutagenesis of a pso-TFO genomic crosslink in repair proficient and deficient cells. Deficiencies in non homologous end joining and mismatch repair do not influence mutation patterns. In contrast, the frequency of base substitutions is dependent on the activity of ERCC1/XPF and polymerase zeta, but independent of other nucleotide excision repair (NER) or transcription coupled repair (TCR) genes. In NER/TCR deficient cells the frequency of deletions rises, indicating that in wild-type cells NER/TCR functions divert pso-TFO crosslinks from processes that result in deletions. We conclude that targeted pso-TFO crosslinks can enter genetically distinct mutational routes that resolve to base substitutions or deletions.

Structural organization, mapping, characterization and evolutionary relationships of CDKN2 gene family members in Xiphophorus fishes.

Xiphophorus fishes and their hybrids are used as models for the study of melanoma and other diseases. The cyclin-dependent kinase inhibitor gene family in humans is comprised of four members, including CDKN2A (P16), and dysregulation of this gene is implicated in numerous neoplasms including melanomas. We have investigated the status of the gene family in the southern platyfish X. maculatus. Xiphophorus harbors at least two such loci, which we now term CDKN2A/B and CDKN2D. Both loci map to Xiphophorus linkage group 5, a genomic area that has long been known to harbor the DIFF tumor suppressor locus. Within this report, we report on the complete cloning, genomic exon/intron boundary delineation, linkage mapping and expressional characteristics of Xiphophorus CDKN2D. We also compare and contrast this expression to that of the previously isolated CDKN2AB locus in normal and neoplastic tissues derived from non-hybrid and hybrid fishes. The hypothetical evolutionary relationships of gene family members and their involvement in melanoma is evaluated. In comparison to CDKN2A/B, the RNA expression of Xiphophorus CDKN2D differs in normal tissues and is not associated with melanotic/pathologic tissues, confirming functional divergence between obvious homologues.