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Title page for ETD etd-12292011-192757


Type of Document Dissertation
Author Guler, Gulfem Dilek
URN etd-12292011-192757
Title Human DNA helicase B in replication fork surveillance and replication stress recovery
Degree PhD
Department Biological Sciences
Advisory Committee
Advisor Name Title
James G. Patton Committee Chair
Daniel Kaplan Committee Member
David Cortez Committee Member
Ellen Fanning Committee Member
Walter J. Chazin Committee Member
Keywords
  • Replication protein A
  • RPA
  • DNA helicase
  • DNA replication
  • DNA repair
  • replication stress
  • HelB
  • HDHB
Date of Defense 2011-12-05
Availability unrestricted
Abstract
Correct and faithful genome duplication is crucial for preserving genomic integrity. Genome duplication is, therefore, highly regulated through a complex network of proteins that accomplish DNA replication in addition to DNA repair, as needed. Human DNA helicase B (HDHB) was previously proposed to function in DNA replication and DNA damage response. Work presented in this dissertation aimed to gain insight into the cellular pathway(s) HDHB participates in, particularly in response to replication stress. Contrary to previous studies where helicase-dead DHB inhibited DNA replication, we did not observe any cell cycle or DNA replication defect in HDHB silenced cells, suggesting that HDHB activity in DNA replication can be compensated by other proteins in the absence of HDHB. On the other hand, HDHB silencing disrupted efficient recovery from replication stress. HDHB silenced cells were capable of activating checkpoint signaling, suggesting a checkpoint-independent or downstream pathway for HDHB function. Consistent with a role in replication stress response, HDHB accumulated on chromatin upon UV, hydroxyurea and camptothecin exposure in a time- and dose-dependent manner. We found that genotoxin-induced HDHB accumulation on chromatin is RPA-dependent. Biophysical and biochemical characterization revealed a direct physical interaction between RPA70N basic cleft and a conserved acidic motif in HDHB helicase domain. The interaction interface between HDHB and RPA70N is strikingly similar to the previously reported interaction interfaces of RPA70N and p53, ATRIP, Rad9 or Mre11. Site-directed mutagenesis of the HDHB-RPA70N interaction interface demonstrated its contribution to HDHB recruitment to chromatin upon genotoxin exposure. These results altogether implicate HDHB in replication stress response.
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