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Influence of Werner helicase deficiency on nonhomologous end‐joining in human cells
Author(s) -
Smith Krissy,
Waldman Alan S.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.878.5
Subject(s) - helicase , non homologous end joining , werner syndrome , biology , microbiology and biotechnology , genetics , plasmid , dna repair , gene , dna , transfection , mutation , premature aging , rna
Mutation of Werner Helicase (WRN) causes Werner Syndrome, a rare disorder associated with symptoms of premature aging and cancer predisposition. WRN has been implicated in DNA double‐strand break (DSB) repair via nonhomologous end‐joining (NHEJ). Plasmid‐based assays suggested that WRN deficiency results in NHEJ that produces large deletions. To our knowledge, the influence of WRN on NHEJ as it occurs in human chromosomes has not been investigated. We stably transfected WRN‐deficient human fibroblast cell line AG11395 with pTNeo99‐7 which contains a thymidine kinase (tk)‐neo fusion gene disrupted by the 18 bp recognition site for endonuclease I‐SceI. A DSB was introduced in the tk‐neo gene by expression of I‐SceI and repair events were recovered by selection for G418‐resistant clones. From AG11395‐derived cell lines, 95 G418‐resistant clones were analyzed and 45 were found to have arisen via simple NHEJ. 39 NHEJ events displayed deletions, while six NHEJ events displayed sequence insertions. Deletions ranged from 1 bp to 619 bp, with a median size of 7 bp. NHEJ in normal human fibroblasts displayed a median deletion size of 37 bp, indicating that WRN deficiency did not increase deletion size. However, nearly half of the DSB repair events recovered from WRN‐deficient cells showed evidence of gross chromosomal rearrangements, suggesting corruption of DSB metabolism. Supported by NIH grant R01GM081472 to ASW.