The Bloom's syndrome (BS) gene, BLM, plays an important role in the maintenance of genomic stability in somatic cells. A candidate for BLM was identified by direct selection of a cDNA derived from a 250 kb segment of the genome to which BLM had been assigned by somatic crossover point mapping. In this novel mapping method, cells were used from persons with BS that had undergone intragenic recombination within BLM. cDNA analysis of the candidate gene identified a 4437 bp cDNA that encodes a 1417 amino acid peptide with homology to the RecQ helicases, a subfamily of DExH box-containing DNA and RNA helicases. The presence of chain-terminating mutations in the candidate gene in persons with BS proved that it was BLM.

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Chromosomal double-strand breaks (DSBs) in mammalian cells are repaired by either homology-directed repair (HDR), using a homologous sequence as a repair template, or nonhomologous end-joining (NHEJ), which often involves sequence alterations at the DSB site. To characterize the interrelationship of these two pathways, we analyzed HDR of a DSB in cells deficient for NHEJ components. We find that the HDR frequency is enhanced in Ku70 Double-strand breaks (DSBs) are potentially catastrophic lesions that if not repaired will lead to loss of genetic information and mutagenesis or cell death. In mammalian cells, two major pathways exist to repair DSBshomologous recombination and nonhomologous endjoining (NHEJ;Liang et al. 1998). NHEJ, the rejoining of DNA ends with the use of little or no sequence homology, involves the processing of ends such that nucleotides are often deleted or inserted at the break site prior to ligation (Jeggo 1998). Such modifications are likely central to the ability of mammalian cells to rejoin DNA ends with a variety of structures. Homology-directed repair (HDR) of a DSB, in contrast, requires significant lengths of sequence homology so that a DNA end from one molecule can invade a homologous sequence and prime repair synthesis (Pâques and Haber 1999). Processing of DNA ends also occurs with HDR; however, repair is typically precise, because a homologous sequence templates the repair event.Several processes exist in which repair of a DSB is restricted to either NHEJ or HDR. For example, DSBs introduced by the RAG proteins to generate antigen receptor diversity during V(D)J rearrangement are repaired by the NHEJ pathway (Jeggo 1998), whereas those introduced during meiosis by the Spo11 protein are repaired by the HDR pathway (Keeney 2001). The restriction in type of DSB repair raises the question as to how pathway choice is regulated. Several studies point to cell cycle phase as one factor that modulates repair pathway choice. In chicken cells, HDR has been found to play a dominant role in repairing radiation-induced DSBs in late S/G 2 phase, whereas NHEJ is preferentially used during G 1 /early S phase (Takata et al. 1998). Consistent with this in mammalian cells, the preferred homologous template for HDR-the sister chromatid (Johnson and Jasin 2001)-is present only during the S/G 2 phase of the cell cycle. Despite a cell cycle preference, HDR and NHEJ can nevertheless be coupled for the repair of a single DSB in mammalian cells (Richardson and Jasin 2000), indicating that the two repair pathways are not completely restricted to different cell cycle phases and that other factors influence pathway choice.Based on in vitro studies of DNA end-binding proteins such as RAD52, it has been suggested that end-binding proteins may direct entry into alternative DSB repair pathways (Van Dyck et al. 1999). However, no evidence yet exists in vivo to support this model, and mutation of the Rad52 gene in mouse does not confer a cellular DSB repair phenotype (Rijkers et al. 1998). NHEJ proteins, that is, th...

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RecQ-like helicase, presumed to function in DNA replication, recombination, or repair. BLM localizes to promyelocytic leukemia protein (PML) nuclear bodies and is expressed during late S and G2. We show, in normal human cells, that the recombination/repair proteins hRAD51 and replication protein (RP)-A assembled with BLM into a fraction of PML bodies during late S/G2. Biochemical experiments suggested that BLM resides in a nuclear matrix–bound complex in which association with hRAD51 may be direct. DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism. This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed. It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci. After radiation, foci containing BLM and PML formed at sites of single-stranded DNA and presumptive repair in normal cells, but not in cells with defective PML. Our findings suggest that BLM is part of a dynamic nuclear matrix–based complex that requires PML and functions during G2 in undamaged cells and recombinational repair after DNA damage.

MMRpro is a broadly applicable, accurate prediction model that can contribute to current screening and genetic counseling practices in a high-risk population. It is more sensitive and more specific than existing clinical guidelines for identifying individuals who may benefit from MMR germline testing. It is applicable to individuals for whom tumor samples are not available and to individuals in whom germline testing finds no mutation.

Diverse functions, including DNA replication, recombination and repair, occur during S phase of the eukaryotic cell cycle. It has been proposed that p53 and BLM help regulate these functions. We show that p53 and BLM accumulated after hydroxyurea (HU) treatment, and physically associated and co-localized with each other and with RAD51 at sites of stalled DNA replication forks. HU-induced relocalization of BLM to RAD51 foci was p53 independent. However, BLM was required for efficient localization of either wild-type or mutated (Ser15Ala) p53 to these foci and for physical association of p53 with RAD51. Loss of BLM and p53 function synergistically enhanced homologous recombination frequency, indicating that they mediated the process by complementary pathways. Loss of p53 further enhanced the rate of spontaneous sister chromatid exchange (SCE) in Bloom syndrome (BS) cells, but not in their BLM-corrected counterpart, indicating that involvement of p53 in regulating spontaneous SCE is BLM dependent. These results indicate that p53 and BLM functionally interact during resolution of stalled DNA replication forks and provide insight into the mechanism of genomic fidelity maintenance by these nuclear proteins.

Bloom syndrome is a rare autosomal disorder characterized by predisposition to cancer and genomic instability. BLM, the structural gene mutated in individuals with the disorder, encodes a DNA helicase belonging to the RecQ family of helicases. These helicases have been established to serve roles in both promoting and preventing recombination. Mounting evidence has implicated a function for BLM during DNA replication; specifically, BLM might be involved in rescuing stalled or collapsed replication forks by a recombination-based mechanism. We have tested this idea by examining the binding and melting activity of BLM on oligonucleotide substrates containing D-loops, DNA structures that model the presumed initial intermediate formed during homologous recombination. We find that BLM preferentially melts those D-loops that are formed more favorably by the strand exchange protein Rad51, but whose polarity could be less favorable for enabling restoration of an active replication fork. We propose a model in which BLM selectively dissociates recombination intermediates likely to be unfavorable for recombination-promoted replication.

We performed a three-phase genome-wide association study (GWAS) using cases and controls from a genetically isolated population, Ashkenazi Jews (AJ), to identify loci associated with breast cancer risk. In the first phase, we compared allele frequencies of 150,080 SNPs in 249 high-risk, BRCA1/2 mutation-negative AJ familial cases and 299 cancer-free AJ controls using 2 and the Cochran-Armitage trend tests. In the second phase, we genotyped 343 SNPs from 123 regions most significantly associated from stage 1, including 4 SNPs from the FGFR2 region, in 950 consecutive AJ breast cancer cases and 979 age-matched AJ controls. We replicated major associations in a third independent set of 243 AJ cases and 187 controls. We obtained a significant allele P value of association with AJ breast cancer in the FGFR2 region (P ‫؍‬ 1.5 ؋ 10 ؊5 , odds ratio (OR) 1.26, 95% confidence interval (CI) 1.13-1.40 at rs1078806 for all phases combined). In addition, we found a risk locus in a region of chromosome 6q22.33 (P ‫؍‬ 2.9 ؋ 10 ؊8 , OR 1.41, 95% CI 1.25-1.59 at rs2180341). Using several SNPs at each implicated locus, we were able to verify associations and impute haplotypes. The major haplotype at the 6q22.33 locus conferred protection from disease, whereas the minor haplotype conferred risk. Candidate genes in the 6q22.33 region include ECHDC1, which encodes a protein involved in mitochondrial fatty acid oxidation, and also RNF146, which encodes a ubiquitin protein ligase, both known pathways in breast cancer pathogenesis.genomics ͉ mapping ͉ disease ͉ predisposition ͉ SNP C ohort and twin studies have indicated that 5-15% of incident breast cancer cases result from autosomal-dominant cancer susceptibility (1-5). However, only Ϸ40% of the familial aggregation of breast cancers can be explained by mutations in BRCA1, BRCA2, or other identified cancer susceptibility genes (6). Attempts to use linkage strategies to localize other genes associated with an inherited predisposition to cancer have been hampered by genetic heterogeneity, decreased penetrance, and chance clustering (7-12). Candidate gene studies in multiplex kindreds affected by breast cancer have implicated rare variants of CHEK2, ATM, BRIP1, and PALB2 in the subset of families lacking BRCA mutations, but in most cases, the rarity and small effect sizes of these associations have precluded clinical application (13). Association studies of biologically plausible candidate genes have identified low-penetrance susceptibility alleles in pathways of carcinogen metabolism, inflammation and immune response, DNA metabolism and DNA repair as well as other known oncogenes and tumor suppressor genes (14-17). Most recently, two groups have carried out genome-wide association studies (GWAS) of selected kindreds and unselected individuals affected by breast cancer (18,19). These studies have implicated a locus near FGFR2 as associated with an Ϸ1.2-fold increased risk of the disease. To add to the potential power of the GWAS approach, we have proposed and validated the use of a genet...