In addition to increased DNA-strand exchange, a cytogenetic feature of cells lacking the RecQ-like BLM helicase is a tendency for telomeres to associate. We also report additional cellular and biochemical evidence for the role of BLM in telomere maintenance. BLM co-localizes and complexes with the telomere repeat protein TRF2 in cells that employ the recombination-mediated mechanism of telomere lengthening known as ALT (alternative lengthening of telomeres). BLM co-localizes with TRF2 in foci actively synthesizing DNA during late S and G2/M; co-localization increases in late S and G2/M when ALT is thought to occur. Additionally, TRF1 and TRF2 interact directly with BLM and regulate BLM unwinding activity in vitro. Whereas TRF2 stimulates BLM unwinding of telomeric and non-telomeric substrates, TRF1 inhibits BLM unwinding of telomeric substrates only. Finally, TRF2 stimulates BLM unwinding with equimolar concentrations of TRF1, but not when TRF1 is added in molar excess. These data suggest a function for BLM in recombination-mediated telomere lengthening and support a model for the coordinated regulation of BLM activity at telomeres by TRF1 and TRF2.

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre-and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.Bloom's syndrome (BS) 1 is a rare autosomal recessive disorder characterized by immunodeficiency, short stature, male infertility, and an increased risk of a broad spectrum of cancers (1). Cells isolated from BS individuals are characterized by cytogenetic abnormalities, with the hallmark feature of hyperrecombination between sister chromatids. BS chromosomes also display increased levels of breaks, translocations, quadriradial formations, and telomeric associations (2).The gene mutated in BS was positionally cloned and named BLM; it encodes a 1417-amino acid protein with strong homology to the Escherichia coli RecQ family of DNA and RNA helicases (3). The E. coli RecQ helicase participates in homologous recombination and suppresses illegitimate recombination (4, 5). Other eukaryotic RecQ family members include Sgs1p from Saccharomyces cerevisiae and Rqh1p from Schizosaccharomyces pombe; loss of function of either of these helicases results in genomic instability (6, 7). Mutations in other human RecQ helicases result in the rare autosomal recessive disorders Werner's syndrome and Rothmund-Thomson syndrome, also characterized by chromosomal instability and cancer predisposition (8, 9).The BLM helicase unwinds duplex DNA from 3Ј to 5Ј in the presence of ATP (10, 11). It also selectively recognizes and promotes branch migration of Holliday junctions in vitro (12). BLM can be found in a large protein complex in the nucleus with other proteins involved in DNA repair such as BRCA1, ATM, MLH1, MSH2, MSH6, and replication factor C (13). However, direct interactions of BLM have only been demonstrated biochemically with replication protein A (RPA) and topoisomerase III␣ (14 -16). These experiments suggest that the BLM helicase interacts with a variety of nuclear proteins to perform functions in DNA replication, recombination, or repair.To understand the role of the BLM helicase in maintaining genomic stability, a yeast two-hybrid screen was used to identify...

Type V collagen is a constituent of type I collagen-rich fibrils in many connective tissues and is a regulator of fibril diameter. In tissues, type V collagen is a heterotrimer with the molecular structure: alpha 1(V)2 alpha 2(V) or alpha 1(V) alpha 2(V) alpha 3(V). We report that genomic polymorphisms at the pro alpha 1(V) gene (COL5A1) locus cosegregated with the gravis form of Ehlers-Danlos syndrome (EDS) (type I) in a three generation family. Affected family members, who had classical features including joint hyperextensibility, fragile skin, and widened, atrophic scars, were heterozygous for a 4 bp deletion at positions from +3 to +6 of intron 65, which resulted in removal of exon 65 sequences from processed mRNAs. Since exon 65 encodes 78 residues of the carboxyl propeptide, the expected result of this mutation is reduced efficiency in incorporating mutant pro alpha 1(V) chains into type V collagen molecules and reduced type V collagen synthesis. These studies indicate that heterozygous mutations in COL5A1 can result in EDS type I. However, linkage studies in other EDS I families indicate the disorder is heterogeneous; linkage to both COL5A1 and COL5A2 was excluded in two other families with EDS I while a fourth family was concordant for linkage to COL5A1 (Z = 2.11; theta = 0.00).

Abstract.The variability of radiation responses in ovarian tumors and tumor-derived cell lines is poorly understood. Since both DNA repair capacity and p53 status can significantly alter radiation sensitivity, we evaluated these factors along with radiation sensitivity in a panel of sporadic human ovarian carcinoma cell lines. We observed a gradation of radiation sensitivity among these sixteen lines, with a five-fold difference in the LD 50 between the most radiosensitive and the most radioresistant cells. The DNA-dependent protein kinase (DNA-PK) is essential for the repair of radiation induced DNA double-strand breaks in human somatic cells. Therefore, we measured gene copy number, expression levels, protein abundance, genomic copy and kinase activity for DNA-PK in all of our cell lines. While there were detectable differences in DNA-PK between the cell lines, there was no clear correlation with any of these differences and radiation sensitivity. In contrast, p53 function as determined by two independent methods, correlated well with radiation sensitivity, indicating p53 mutant ovarian cancer cells are typically radioresistant relative to p53 wild-type lines. These data suggest that the activity of regulatory molecules such as p53 may be better indicators of radiation sensitivity than DNA repair enzymes such as DNA-PK in ovarian cancer.