Oncogene (2006) 25, 1612–1619 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc REVIEW Mouse models of XRCC1 DNA repair polymorphisms and cancer WC Ladiges Department of Comparative Medicine, University of Washington, Seattle, WA, USA DNA damage plays a major role in mutagenesis, that plays a major role in many cases of cancer. carcinogenesis and aging. A gene that is emerging as an Oxidatively modified DNA is present in many tissues essential element in the repair of both damaged bases and including tumor tissue (reviewed by De Bont and van single-strand breaks (SSB) is XRCC1. XRCC1 has been Larebeke, 2004). Oxidative stress occurs when the shown to have a large number of single-nucleotide production of ROS exceeds the body’s natural defense polymorphisms (SNPs),several of which are being mechanisms, causing damage to macromolecules such as increasingly studied in cancer epidemiology investigations, DNA. Any change in the efficacy of DNA repair will in part because of their relative high frequency in the alter the steady-state levels of oxidative DNA modifica- population. Although association trends with specific tions, which in turn affects the mutation rate and cancer types have occasionally been shown in a variety ultimately the cancer incidence. Oxidative base damage of ethnic backgrounds,there are often conflicting reports and single-strand breaks (SSB) are the most frequent that weaken any substantial conclusions. The functional types of DNA damage caused by ROS (Thompson and significance of these SNPs is still largely unknown. West, 2000), and if not repaired, can lead to much more XRCC1 is an excellent prototype to provide a forum for serious double-strand breaks that directly contribute to determining how epidemiological cancer association stu- the development of cancer. There is thus a great dies with DNA repair gene polymorphisms can be appreciation for molecular systems designed to repair validated or refuted. The focus is on the utilization of in damaged DNA. Base damage is handled by a process silico data and biochemical studies in cell lines and termed base excision repair, which consists of removal existing mouse models to help provide a framework for the by glycosylases followed by processing by XRCC1- development of new mutant mouse lines that mimic human assisted POL b-dependent short-patch and/or prolifer- polymorphisms. These mouse lines will provide the next ating cell nuclear antigen/POL b-dependent long-patch generation of mammalian tools for carcinogen exposure repair. SSB are repaired by activation of poly (ADP- studies relevant to human cancer and variations in ribose) polymerases (PARPs) and XRCC1, with sub- XRCC1,and provide the basis for investigating groups sequent recruitment of additional proteins involved in of genes and polymorphisms in an animal model. the repair process. A gene that is emerging as an Oncogene (2006) 25, 1612–1619. doi:10.1038/sj.onc.1209370 essential element in the repair of both damaged bases and SSB is XRCC1 (Figure 1). Keywords: DNA repair; polymorphisms; XRCC1; XRCC1 was the first human gene involved in SSB cancer; mouse models repair to be cloned (reviewed by Thompson and West, 2000). Cells lacking the gene product are hypersensitive to ionizing radiation, hydrogen peroxide, camptothecin and alkylating agents (reviewed by Caldecott, 2003). In addition, mutant cells show elevated frequency of Introduction spontaneous chromosomal aberrations and deletions, and null mutant mice exhibit an embryonic lethal DNA damage plays a major role in mutagenesis, phenotype indicating the importance of XRCC1 for carcinogenesis and aging. There are a number of genetic stability (Tebbs et al., 1999). A recent study has chemical events that lead to DNA damage including shown that XRCC1 is required for efficient DNA SSB hydrolysis, exposure to reactive oxygen species (ROS) repair (Brem and Hall, 2005). Interestingly, XRCC1 has and other reactive metabolites. These events result from been shown to have a large number of single-nucleotide metabolic, endogenous processes, or are triggered by polymorphisms (SNPs), several of which are being exposure to exogenous chemicals. It is now becoming increasingly studied in cancer epidemiology investiga- apparent that mutations due to DNA damage are tions, in part because of their relative high frequency in caused by endogenous factors that are modulated by the population. Although association trends with exogenous factors, and it is probably this combination specific cancer types have occasionally been shown in a variety of ethnic backgrounds, there are often conflicting reports that weaken any substantial conclu- Correspondence: Dr WC Ladiges, Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA. sions. Consequently, the functional significance of these E-mail: [email protected] SNPs is still largely unknown. This paper will use XRCC1 mouse models WC Ladiges 1613 XRCC1 as a prototype to provide a forum for how known enzymatic activity has been attributed to epidemiological cancer association studies with DNA XRCC1, but three interactive domains have been repair gene polymorphisms can be validated or refuted. identified (Figure 2), plus a nuclear localization signal The focus will be on the utilization of in silico data and and a phosphorylation site for Ck2 (Loizou et al., 2004). biochemical studies in cell lines and existing mouse The N-terminal domain (NTD) is the site for POL models to help provide a framework for the develop- b binding (Dianova et al., 2004). It is also the site for ment of new mutant mouse lines that mimic human direct binding to both gapped and nicked DNA, and polymorphisms. These mouse lines will provide the next gapped DNA complexed with POL b (Marintchev et al., generation of mammalian tools for environmental 1999). XRCC1 has two BRCT domains, which are exposure studies relevant to human cancer. weakly conserved motifs first identified in BRCA1, and which mediate protein interactions (Zhang et al., 1998; Taylor et al., 2002; Beernink et al., 2005). BRCT1 is an XRCC1 structure interactive site for PARP whereas BRCT2 is an interactive binding site for Lig3. The linker area between Human XRCC1 is composed of 17 exons and spans a the NTD and BRCT1 has been suggested to be the area genomic distance of 32 kb (Thompson and West, 2000). for binding with other proteins including Apex, OGG1 It is located on chromosome 19q 32–133. Mouse (Marsin et al., 2003) and PCNA (Fan et al., 2004). XRCC1 maps to a similar region on chromosome 7 and spans 26 Kb. The exon positions are the same for mouse and human, and there is a high degree of XRCC1 polymorphisms and cancer association studies homology of amino-acid residues (MGI database). No An extensive review of the epidemiology will not be presented, as there are several published documents that address this issue very well. Rather, an attempt will be made to speculate on association trends and how this Carcinogen information can be used to make decisions for investigating functional aspects of the XRCC1 gene. The Nickerson lab reports 27 gene variations for XRCC1 using a panel of 90 human lymphoid cell lines Oxidative stress (http://egp.gs.washington.edu/). Of these, three are 50 UTR’s, 12 are insertion/deletions, four are synon- Antioxidant enzymes ymous SNPs, and eight are nonsynonymous SNPs (Figure 3). Only two polymorphisms have been studied DNA in any detail because they occur with a high frequency in damage DNA single strand break the population. R399Q is most frequent followed by R194W. This frequency allows lower cohort numbers to POLβ be studied thus providing OR data that can be analyzed Parp to a significant level. A third polymorphism, R280H, has Lig3 also been studied but less frequently because of its lower prevalence in the population. Ck2 XRCC1 Figure 1 Schematic overview of the response of XRCC1 and its R194W interactive protein partners to a DNA single-strand break induced A number of cancer association studies have been by an environmental carcinogen and oxidative stress. reported. Based on a recent review by Goode et al. NTD NLS Brct1 Ck2 Brct2 84-183 183-310. PCNA, Ape 315-403 514-530 538-629 POLβ Ogg1, MutyH? Parp1 phosphorylation Lig3 Parp2 PNK Figure 2 Human XRCC1 domains and locations of binding sites with interactive protein partners. Oncogene XRCC1 mouse models WC Ladiges 1614 NTD NLS Brct1 Ck2 Brct2 P161L R399Q T576S P309S V72H R194W T304A R280H Figure 3 Human XRCC1 domains and locations of the eight nonsynonymous (change in amino-acid residue) single-nucleotide polymorphisms (SNP) as identified by the Nickerson lab (http://egp.gs.washington.edu). (2002), six studies reported a reduced risk of cancer hemizygosity of the XRCC1 gene in CHO cells accounts associated with the R194W SNP. These were all case for the relative ease of isolating mutants with pheno- control studies with each having greater than 200 cases. types of sensitivity to agents, such as methyl methane- This association was confirmed by a more recent meta- sulfonate (MMS), that produce high levels of SSB. One analysis of 38 case control studies in Chinese popula- mutant clone, designated as EM9, was shown to carry a tions (Hu et al., 2005). A Russian study of more than frameshift mutation that results in truncated polypep- 2000 cases reported that R194W was associated with a tide lacking two-thirds of the normal sequence. EM9 is reduced risk of lung cancer among subjects in the thus a null mutant with genetic characteristics compar- highest quartile of pack-years of smoking (Hung et al., able to mouse cells carrying a knockout mutation. 2005). These results are somewhat surprising since a Takanami et al. (2005) made plasmid constructs common perception is that a change in amino-acid expressing XRCC1 wild-type, R194W or R280H and structure would be deleterious to function and result in transfected them into EM9 CHO cells.