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Title page for ETD etd-07162011-113100

Type of Document Master's Thesis
Author Alers Rivera, Ileana
Author's Email Address ileana.alers@vanderbilt.edu
URN etd-07162011-113100
Title NMR analysis of the RPA dimer core RPA32D/14
Degree Master of Science
Department Chemical and Physical Biology
Advisory Committee
Advisor Name Title
Walter J. Chazin Committee Chair
Albert H. Beth Committee Member
Borden D. Lacy Committee Member
  • telomeres
  • Replication Protein A
  • G-quadruplex
Date of Defense 2011-06-28
Availability unrestricted



Thesis under the direction of Professor Walter J. Chazin

Replication protein A (RPA) is the primary eukaryotic single-stranded DNA binding protein. RPA is an essential factor for DNA transactions including replication, repair and telomere maintenance, when the DNA is unwound for processing. Binding of RPA protects ssDNA from nucleases, formation of secondary structures and reannealing of the two strands. RPA also serves as a scaffold that orchestrates the assembly and disassembly of DNA processing machinery. RPA is a heterotrimer of subunits RPA70, RPA32 and RPA14, which together contain six OB-fold, one winged-helix and one disordered domain. Characterization by NMR of the structure and dynamics of RPA and the effect of interactions with DNA and DNA processing proteins is being used to understand the physical basis for RPA function. Recently, RPA was shown to bind DNA G-quadruplexes found at the end of telomeres and to disrupt these structures. This activity has been mapped to the RPA32/14 dimer. To enable NMR analysis of this system, we have undertaken a series of three-dimensional triple resonance NMR experiments acquired with TROSY enhancement at 800 MHz to assign the backbone resonances of RPA32D/14. Assignments were made for approximately 60% of the 149 residues of RPA32D/14 from these data and about half were confirmed in a NOESY experiment. These resonance assignments lay a foundation for characterizing the interaction of RPA32D/14 with G-quadruplex structures, and will facilitate future NMR analysis of the RPA trimer core and the full-length protein.

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