title: Depletion of the bloom syndrome helicase stimulates homology-dependent repair at double-strand breaks in human chromosomes.
abstract: Mutation of BLM helicase causes Blooms syndrome, a disorder associated with genome instability, high levels of sister chromatid exchanges, and cancer predisposition. To study the influence of BLM on double-strand break (DSB) repair in human chromosomes, we stably transfected a normal human cell line with a DNA substrate that contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI. The substrate also contained a closely linked functional tk gene to serve as a recombination partner for the tk-neo fusion gene. We derived two cell lines each containing a single integrated copy of the DNA substrate. In these cell lines, a DSB was introduced within the tk-neo fusion gene by expression of I-SceI. DSB repair events that occurred via homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recovered by selection for G418-resistant clones. DSB repair was examined under conditions of either normal BLM expression or reduced BLM expression brought about by RNA interference. We report that BLM knockdown in both cell lines specifically increased the frequency of HR events that produced deletions by crossovers or single-strand annealing while leaving the frequency of gene conversions unchanged or reduced. We observed no change in the accuracy of individual HR events and no substantial alteration of the nature of individual NHEJ events when BLM expression was reduced. Our work provides the first direct evidence that BLM influences DSB repair pathway choice in human chromosomes and suggests that BLM deficiency can engender genomic instability by provoking an increased frequency of HR events of a potentially deleterious nature.
authors.affiliation: Department of Biological Science, University of South Carolina, 700 Sumter Street, Columbia, SC 29208, USA. yibinw@biol.sc.edu
authors.forename: Yibin
authors.initials: Y
authors.lastname: Wang
authors.forename: Krissy
authors.initials: K
authors.lastname: Smith
authors.forename: Barbara Criscuolo
authors.initials: BC
authors.lastname: Waldman
authors.forename: Alan S
authors.initials: AS
authors.lastname: Waldman
mesh_terms: D001483:Base Sequence; D001816:Bloom Syndrome / Q000201:enzymology* / Q000235:genetics*; D002460:Cell Line; D002877:Chromosomes, Human; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair; D055785:Gene Knockdown Techniques; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D016415:Sequence Alignment
chemical_list: D034741:RNA, Small Interfering; D004265:DNA Helicases