BRCA1 is a susceptibility gene for breast and ovarian cancer with growth-inhibitory activity for which the mechanism of action remains unclear. When introduced into cells, BRCA1 inhibits growth of some but not all cell lines. In an attempt to uncover the mechanism of growth suppression by BRCA1, we examined a panel of cell lines for their ability to reduce colony outgrowth in response to BRCA1 overexpression. Of all variables tested, only those cells with wild-type pRb were sensitive to BRCA1-induced growth suppression. In cells with an intact rb gene, inactivation of pRb by HPV E7 abrogates the growth arrest imposed by BRCA1. In accordance with these observations, we found that BRCA1 could not suppress BrdUrd uptake in primary fibroblasts from rb؊/؊ mice and exhibited an intermediate ability to inhibit DNA synthesis in rb؉/؊ as compared with rb؉/؉ cells. We further found that the BRCA1 protein complexes with the hypophosphorylated form of pRb. This binding is localized to amino acids 304 -394 of BRCA1 protein and requires the ABC domain of pRb. In-frame deletion of BRCA1 fragment involved in interaction with pRb completely abolished the growth-suppressive property of BRCA1. Although it has been reported that BRCA1 interacts with p53, we find the p53 status did not affect the ability of BRCA1 to suppress colony formation. Our data suggest that the growth suppressor function of BRCA1 depends, at least in part, on Rb. G ermline mutations in BRCA1 are found in Ϸ50-60% of hereditary breast and ovarian cancers (1). Despite the unequivocal role of BRCA1 in familial breast cancer susceptibility, the biological function of the BRCA1 protein remains unclear. Experimental data suggest that BRCA1 may be a negative regulator of cell growth. Attenuation of BRCA1 synthesis by antisense oligonucleotide increased the proliferative rate of both benign and malignant mammary epithelial cells in culture (2), and BRCA1 expression decreased the capacity of MCF7 breast cancer cells to form tumors in nude mice (3). However, BRCA1 expression increases as cells progress through the G 1 and S phases of the cell cycle (4, 5). Furthermore, homozygous BRCA1 mutant mice died at day 7.5 of embryogenesis with evidence of abnormal cessation of cellular proliferation accompanied by high levels of the CDK inhibitor p21 and low levels of cyclin E and mdm2 (6, 7). Recently, colocalization and physical interaction of BRCA1 with Rad51 protein has raised the possibility that BRCA1 is involved in DNA repair (8). Moreover, the COOH terminus of the BRCA1 protein can activate transcription in in vitro experiments (9, 10) and coactivate transcription of p53-regulated genes (11,12).The experimental biology of BRCA1 therefore suggests that BRCA1 may have different functions, each best manifested in specific study systems and cell lines. Herein, we show that BRCA1 binds preferentially to the hypophosphorylated form of Rb and that the growth-suppressive phenotype of BRCA1 depends on the presence of a functional Rb protein. These data indicate the complexity of ...
The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5 to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICLspecific incision on the 3-side of an ICL. The ICL-specific 3-incision, along with the 5-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork. DNA interstrand cross-links (ICLs)2 are formed as a result of DNA damage to each strand of the duplex. These lesions are located one or two nucleotides apart on the opposite strands and covalently link them, blocking DNA replication and transcription (1-3). Therefore, ICLs are considered to be the most cytotoxic DNA lesion (2-4).Several models for mammalian ICL repair have been proposed (2, 5-7). The first critical step for repairing ICLs in any given model is to release the ICL from one of the strands by making two incisions that bracket an ICL (unhooking the ICL). The unhooking of ICLs permits strand separation. In Escherichia coli and yeast, nucleotide excision repair (NER) makes dual incisions bracketing the ICL on one of the cross-linked strands to unhook ICLs (2, 3). Interestingly, NER is dispensable in mammalian ICL repair (5,8). In contrast to NER-defective mutants in E. coli and yeast, the NER-defective mammalian cells (except for ERCC-1 and ERCC-4 cells) are only moderately sensitive to DNA cross-linking agents such as mitomycin C (MMC) (8 -11). Moreover, biochemical studies demonstrated that the dual incisions by mammalian NER do not "unhook" ICLs (12, 13). Therefore, mammalian cells evidently have a unique mechanism(s) to initiate ICL repair.Notably, double strand break (DSB) repair-defective mutant cells, including those deficient in Rad51 paralogs or BRCA2, are hypersensitive to DNA cross-linking agents such as MMC (10). It has also been reported that ICLs in the mammalian genome are removed during S-phase and the DNA replication-mediated formation of a DSB is the key intermediate in ICL repair in mammalian cells (1,8,14,15). These data indicate that mammalian ICL repair goes through three critical processes: the formation of DNA replication-mediated DSBs, unhooking of the ICLs (separation of the two linked strands), and repair of the DSBs (restoration of the collapsed replication fork). It is unknown whether the formation of DSBs precedes the unhooking reaction. When the DNA replication complex encounters an ICL, the strand separat...
Human DNA polymerase N (PolN) is an A-family nuclear DNA polymerase whose function is unknown. This study examines the possible role of PolN in DNA repair in human cells treated with PolN-targeted siRNA. HeLa cells with siRNA-mediated knockdown of PolN were more sensitive than control cells to DNA cross-linking agent mitomycin C (MMC), but were not hyper-sensitive to UV irradiation. The MMC hyper-sensitivity of PolN knockdown cells was rescued by the overexpression of DNA polymerase-proficient PolN but not by DNA polymerase-deficient PolN. Furthermore, in vitro experiments showed that purified PolN conducts low efficiency non-mutagenic bypass of a psoralen DNA interstrand cross-link (ICL), whose structure resembles an intermediate in the proposed pathway of ICL repair. These results suggest that PolN might play a role in translesion DNA synthesis during ICL repair in human cells. KeywordsDNA interstrand cross-links; DNA polymerase N; TLS; Psoralen; Chemotherapeutics E. coli PolI is a high fidelity DNA repair polymerase that conducts gap-filling DNA synthesis during nucleotide excision repair (NER), base excision repair (BER), and DNA interstrand cross-link (ICL) repair. E. coli PolI is the prototypical member of A-family DNA polymerases (1). Drosophila melanogaster Mus308 is an A-family nuclear DNA polymerase, the mutants of which are hyper-sensitive to DNA cross-linking agents but not to other DNA damaging agents (2)(3)(4). This suggests that Mus308 may play a role in ICL repair in Drosophila. Two nuclear A-family DNA polymerases, DNA polymerase N (PolN) and DNA polymerase Q (PolQ) were recently discovered (5,6). It has been proposed that PolN and PolQ are mammalian orthologs of Mus308 and that they participate in ICL repair in mammalian cells. However, additional studies are needed to confirm the precise role(s) of PolN and PolQ in DNA repair in mammalian cells.ICLs are generated endogenously as bi-functional products of lipid peroxidation and by exogenous exposure to DNA damaging agents, some of which are commonly used as cancer chemotherapeutic drugs (7,8). Because ICLs covalently link the two complementary strands of duplex DNA, they prevent progression of the DNA replication fork and block RNA transcription. This property makes ICLs highly toxic to proliferating cells. The molecular mechanism of human ICL repair is poorly understood (7-10). The current model of mammalian ICL repair (7-10) suggests that when a DNA replication fork stalls at an ICL, it is recognized *To whom correspondence should be addressed. tbessho@unmc by an endonuclease that generates a double-strand break (DSB) in the vicinity of the ICL (11)(12)(13)(14). Subsequently, XPF-ERCC1 unhooks the ICL, resulting in a gap across from the unhooked ICL (14-17), which cannot be sealed by replicative DNA polymerases δ or ε. Gaps generated during ICL repair are thought to be repaired by translesion DNA synthesis (TLS) or by homologous recombination using the homologous chromosome (the sister chromatid is not available). Once the gap is sea...
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