Localization of the Bloom syndrome helicase to punctate nuclear structures and the nuclear matrix and regulation during the cell cycle: comparison with the Werner's syndrome helicase.
The Bloom (BLM) and Werner's (WRN) syndrome proteins may regulate recombination and DNA repair. Using a novel polyclonal antibody to human BLM, we detected the 170-kda BLM antigen in wild-type but not Bloom syndrome cells. BLM was localized to punctate nuclear structures. The level of BLM but not WRN was 3.6 fold-higher in G(1)/S-synchronized fibroblasts than in G(0)-synchronized fibroblasts. BLM-positive cells invariably expressed topoisomerase IIalpha, whereas topoisomerase IIbeta was expressed constitutively. Transfections of BLM deletion mutants demonstrated that the C-terminal domain of BLM mediated nuclear entry and the central helicase domain was necessary for producing the punctate pattern. By subcellular fractionation, BLM was found primarily in high-salt extracts of the nucleoplasm and the nuclear matrix and was enriched in G(1)/S-synchronized cells compared with G(0)-synchronized cells. There was no interaction between BLM and WRN or topoisomerases IIalpha and IIbeta in fibroblasts. These results demonstrate that BLM is targeted to specific nuclear structures and that its expression is enhanced during cell growth. The known nucleolar localization of WRN, its invariant expression during the cell cycle, and the lack of interaction between BLM and WRN suggest distinct roles for BLM and WRN in processes such as DNA repair and recombination.
Gharibyan
V
V
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
Youssoufian
H
H
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001402:B-Lymphocytes; D001816:Bloom Syndrome / Q000201:enzymology* / Q000235:genetics; D002453:Cell Cycle; D002467:Cell Nucleus / Q000201:enzymology*; D002478:Cells, Cultured; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D015530:Nuclear Matrix / Q000201:enzymology*; D053484:RecQ Helicases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D012867:Skin / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014898:Werner Syndrome / Q000201:enzymology* / Q000235:genetics; D000071657:Werner Syndrome Helicase
D011994:Recombinant Proteins; D005090:Exodeoxyribonucleases; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase