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Title page for ETD etd-09152007-110210


Type of Document Dissertation
Author Liu, Hanjian
Author's Email Address hanjian.liu@vanderbilt.edu
URN etd-09152007-110210
Title Human DNA helicase B functions in DNA damage response and homologous recombination
Degree PhD
Department Biological Sciences
Advisory Committee
Advisor Name Title
James Patton Committee Chair
David Cortez Committee Member
Ellen Fanning Committee Member
Eugene Oltz Committee Member
Jennifer Pietenpol Committee Member
Keywords
  • chromatin association
  • DNA damage response
  • helicase
  • homologous recombination
  • ATR
  • DNA repair
Date of Defense 2007-05-23
Availability unrestricted
Abstract
DNA damaging agents have been shown to stimulate the localization of human DNA helicase B (HDHB) in nuclear foci, suggesting that HDHB might participate in DNA damage response. In the first part of this dissertation, we found that the chromatin-associated fraction of HDHB increases in cells exposed to a variety of DNA damaging agents. HDHB chromatin accumulation is most prominent in S phase cells exposed to agents that cause replication fork stalling or collapse. Inhibition of checkpoint kinases does not prevent damage-induced accumulation of HDHB on chromatin, suggesting that HDHB associates directly with DNA lesions or with other proteins recruited to lesions. Silencing of HDHB does not affect UV-induced RPA focus formation, but diminishes induction of TopBP1 foci. DNA damage induced CHK1 phosphorylation is impaired in HDHB-depleted cells. These results identify HDHB as a novel factor that associates with damaged chromatin and promotes intra-S phase damage responses.

In the second part of this dissertation, we further investigated the possible function of HDHB in homologous recombination. HDHB-depleted cells showed more aphidicolin-induced chromosome breaks than control-depleted cells. HDHB-depleted cells have fewer sister chromatid exchange than control-depleted cells. An in vivo recombination assay showed that HDHB silencing results in impaired homologous recombination. In vitro, recombinant HDHB stimulates Rad51-mediated 5-3 heteroduplex extension. Our studies suggest a function of HDHB in stimulating ssDNA/duplex structure during homologous recombination.

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