Oncogene (2003) 22, 5784–5791 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Roles of BRCA1 and BRCA2 in homologous recombination, DNA replication fidelity and the cellular response to ionizing radiation Simon N Powell*,1 and Lisa A Kachnic2 1Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA, USA; 2Department of Radiation Oncology, Boston Medical Center, Boston, MA, USA Inheritance of one defective copy of either of the two Identification of the BRCA genes breast cancer susceptibility genes, BRCA1 and BRCA2, predisposes individuals to breast and ovarian cancers. The genetic basis for familial breast cancer predisposi- Current progress in determining the function of these tion has become established over the past decade with genes suggests that they participate in a common pathway the cloning of two major breast cancer susceptibility to facilitate orderly homologous recombination and genes, BRCA1 and BRCA2 (Miki et al., 1994; Wooster thereby maintain genomic integrity. As a consequence of et al., 1995). The BRCA1 gene was first cloned in 1994 this defect in homologous recombination, tumors that after being mapped to chromosome 17q21 by genetic arise in BRCA carriers are likely to be more sensitive to linkage analysis. The BRCA1 protein consists of 1863 ionizing radiation. This review summarizes recent inves- amino acids and is expressed in most proliferating cells tigations about the nature of the defect in DNA repair, (Figure 1). Within the cell, BRCA1 is part of a large and highlights the unanswered questions about the tumor protein complex up to 3 MDa in size. The C-terminus suppressor paradox of BRCA genes. The unsolved of BRCA1 contains an amino-acid sequence motif, mystery is the other genetic changes that must occur to now known as a BRCT domain, recognized in many turn a BRCA-deficient cell from a nonviable cell into a DNA repair proteins. This appears to be a site of tumor cell capable of endless growth. protein–protein interactions, perhaps with other DNA Oncogene (2003) 22, 5784–5791. doi:10.1038/sj.onc.1206678 repair proteins to build a large protein complex. In the N-terminus, there is a ring-finger domain, also allowing Keywords: breast cancer; BRCA genes; tumor suppres- for protein–protein interactions, but thought to be sor genes; DNA repair; ionizing radiation; homologous involved in protein ubiquitination. The BRCA2 gene, recombination; DNA replication a very large protein mapped to chromosome 13q12-13, contains 3418 amino acids and encodes a protein of approximately 385 kDa molecular weight (Figure 1). BRCA2 is characterized by a very large exon 11, which Introduction encodes peptide motifs required for interaction with the RAD51 protein (Chen et al., 1999b). The contribution of classical human genetics was Together, mutations in BRCA1 and BRCA2 account paramount in the identification of two breast cancer for the majority of families with hereditary susceptibility susceptibility genes, BRCA1 and BRCA2. Current to breast and ovarian cancer (Hall et al., 1990; Hofmann progress in determining the function of these genes and Schlag, 2000; Paakkonen et al., 2001). Carriers of suggests that they participate in a common pathway that the BRCA gene mutations are heterozygous (one is involved in the control of DNA replication fidelity normal copy and one mutated copy) within the germ and DNA damage repair. Familial breast and ovarian line. Recent epidemiologic studies indicate that BRCA1 cancer can be triggered by a mutation in one allele of mutation carriers have a high lifetime risk of breast either BRCA1 or BRCA2. The sequence of events that cancer of up to 80% (Satagopan et al., 2001). The breast occur from the initial germline mutation to the cancer risk for the BRCA2 mutation carriers approaches development of cancer is unclear, but loss of function that of BRCA1, however, with a later age of disease of the second allele is required. However, if both alleles onset (Schubert et al., 1997). In addition to the breast are knocked out, there is failure to develop or cancer risk, women with BRCA1 mutations have an proliferate. Solving this ‘tumor suppressor paradox’ increased risk of ovarian cancer (Ford et al., 1994). and clarifying the function of the BRCA genes may BRCA2 carriers are also at an increased risk for provide further insight on how inactivation leads to developing ovarian cancer as well as other solid tumors: tumorigenesis. prostatic, pancreatico-biliary, gastric and colon cancers, as well as melanoma (Peto et al., 1999). The BRCA2 mutation appears to confer higher risks for male breast *Correspondence: SN Powell, Department of Radiation Oncology, cancer (Ford et al., 1998). There are subtle histologic Massachusetts General Hospital, Cox 302, 100 Blossom Street, differences between BRCA1- and BRCA2-linked breast Boston, MA 02114, USA; E-mail: [email protected] cancers (Lakhani et al., 1998). The breast cancers that BRCA1 and BRCA2 in DNA replication fidelity SN Powell et al 5785 classified in BRCA-associated murine breast cancers, which failed to suppress transformation but retained other wild-type functions of P53 (Smith et al., 1999a). Additionally, P53 inactivation partially rescued the developmental arrest in BRCA1 and BRCA2 null mice, further supporting the role of the BRCA genes in DNA damage repair and the hypothesis that P53 checkpoint loss is associated with BRCA gene loss in cancer development (Jacks and Weinberg, 1998). The BRCA tumor suppressor paradox It is generally accepted that a cancer results from the Figure 1 A schematic structure of BRCA1 and BRCA2, showing accumulation of genetic changes in a target cell. More the key features and domains of both proteins. They are unrelated than 30 years ago, Knudson hypothesized that carcino- proteins. BRCA1 is characterized by the BRCT motif at the C- genesis results from the occurrence of a second mutation terminus, the SQ cluster region (the site of phosphorylation by ATM and ATR) and the ring domain in the N-terminus (with E3 in a somatic target cell, so that the difference between ubiquitin ligase structure). BRCA2 is characterized by the very hereditary and nonhereditary cancers is the timing of the large exon 11, containing the eight BRC peptide motifs, important first mutation (prezygotic vs postzygotic). The second for the interaction with RAD51. A ssDNA binding domain is mutation can affect the remaining normal allele of the found within the C-terminus of BRCA2 (Figure 1) same gene (recessive trait) or one copy of a different gene (dominant trait). The first evidence substantiating arise in BRCA1 carriers tend to have distinctive patho- this ‘two-hit’ hypothesis or recessive model was the iden- logical features: invasive ductal, high grade, abundant tification of the retinoblastoma tumor-suppressor gene. lymphocyte infiltration, and usually estrogen receptor Breast cancer represents an exception to this two-hit negative (Lakhani et al., 2000). In contrast, tumors model, as it does not show the predicted relation arising in BRCA2 carriers do not differ significantly between hereditary and nonhereditary tumorigenesis. from noncarriers (Lakhani et al., 2002). Many aspects of Tumorigenesis in individuals with germline BRCA the function of BRCA1 and BRCA2 suggest a common mutations requires inactivation of the remaining wild- pathway of DNA recombinational repair; so the reasons type allele, suggesting that the BRCA genes are tumor- for this subtle difference in pathology are not clear. suppressor genes. However, unlike other tumor sup- pressor genes, no disease-causing dominant BRCA1 or BRCA2 mutations have been detected in sporadic breast Knockout mice or ovarian cancers. Epigenetic regulation of the expres- BRCA1 and BRCA2 null embryos (loss of both functional sion of BRCA1 has been reported in human breast alleles) die early in development (approximately day 8 cancers, especially in high-grade tumors (Wilson et al., or 9), with a severe growth deficit. The loss of BRCA gene 1999). There remains the possibility that a BRCA- function in embryogenesis has been shown to cause dependent ‘pathway’ can be inactivated not just by loss an elevated expression of the cell cycle inhibitor, P21, of BRCA1 or BRCA2, but by other components of the and subsequent growth arrest (Hakem et al., 1996, 1998; BRCA-regulated response network, and these may play Suzuki et al., 1997). The P53-dependent growth arrest a role in sporadic breast cancer. associated with a deficiency in BRCA proteins may The ‘caretaker’ gene model of Kinzler and Vogelstein represent a checkpoint response to DNA damage. Con- (1997) could explain this BRCA tumor-suppressor versely, loss of such DNA-damage responsive checkpoint paradox. The inactivation of a caretaker gene may function may collaborate with loss of BRCA-mediated indirectly promote cancer by causing genomic instabil- function in the development of tumors. Additional ity. This hypothesis would suggest that mutations in observations suggest that the embryos arising in BRCA1 BRCA1 or BRCA2 might predispose to cancer develop- or BRCA2 knockouts are radiation sensitive, as observed ment, and the loss of other genes that cooperate with the by apoptosis of the inner cell mass following exposure to loss of BRCA function is necessary for the full 8 Gy of ionizing radiation (Sharan et al., 1997). development of cancer. Whether heterozygosity at the In the clinical setting, breast cancers that arise in loci for the BRCA genes is associated with a haplo- carriers of BRCA1 or BRCA2 mutations are associated insufficiency state is not yet clear. The next step from with a high incidence of inactivation of P53, perhaps germline heterozygosity to cancer development is critical greater than 90% (Crook et al., 1997, 1998; Smith et al., to understanding the function of these genes in vivo. 1999b). In the mouse model, when BRCA1 gene inactivation was achieved with the use of a CRE-lox Response to ionizing radiation vector system (Xu et al., 1999a), the mice developed late onset breast cancer with frequent P53 mutations.