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Vol. 15, Issue 7, 3320-3332, July 2004

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Cell Cycle-dependent Regulation of a Human DNA Helicase That Localizes in DNA Damage Foci

Jinming Gu ^* , Xiaobo Xia  , Peijun Yan ^* , Hanjian Liu ^* , Vladimir N. Podust ^* , Albert B. Reynolds  , and Ellen Fanning ^*  

^* Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235; Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232

Submitted March 17, 2004; Revised April 27, 2004; Accepted April 29, 2004
Monitoring Editor: Keith Yamamoto

Mutational studies of human DNA helicase B (HDHB) have suggested that its activity is critical for the G1/S transition of the cell cycle, but the nature of its role remains unknown. In this study, we show that during G1, ectopically expressed HDHB localizes in nuclear foci induced by DNA damaging agents and that this focal pattern requires active HDHB. During S and G2/M, HDHB localizes primarily in the cytoplasm. A carboxy-terminal domain from HDHB confers cell cycle-dependent localization, but not the focal pattern, to a reporter protein. A cluster of potential cyclin-dependent kinase phosphorylation sites in this domain was modified at the G1/S transition and maintained through G2/M of the cell cycle in vivo, coincident with nuclear export of HDHB. Serine 967 of HDHB was the major site phosphorylated in vivo and in vitro by cyclin-dependent kinases. Mutational analysis demonstrated that phosphorylation of serine 967 is crucial in regulating the subcellular localization of ectopically expressed HDHB. We propose that the helicase of HDHB operates primarily during G1 to process endogenous DNA damage before the G1/S transition, and it is largely sequestered in the cytoplasm during S/G2.

Abbreviations used: Gal, -galactosidase; -PPase, -phosphatase; CDK, cyclin dependent kinase; CRM1, chromosome region maintenance 1; GFP, green fluorescent protein; HDHB, human DNA helicase B; LMB, leptomycin B; Mut, mutant; NES, nuclear export signal; NHEJ, non-homologous end-joining; NLS, nuclear localization signal; PVDF, polyvinylidene difluoride; PSLD, phosphorylation and subcellular localization domain; SLD, subcellular localization domain.

Corresponding author. E-mail address: ellen.fanning{at}vanderbilt.edu

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