Mark C. Williams
Paul M. Champion, Nathan E. (Nathan Eli) Israeloff (1961-)
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Arts and Sciences. Department of Physics.
Biophysics DNA stretching, Nucleocapsid protein, Optical tweezers
DNA-protein interactions, RNA-protein interactions, Molecular spectroscopy, Nucleic acids, DNA, Proteins
The nucleocapsid protein (NC) plays an important role in retroviral replication, in part, by facilitating numerous nucleic acid rearrangements throughout the reverse transcription process. The nucleic acid chaperone activity of the human immunodeficiency virus type-1 (HIV-1) NC has been extensively studied, and duplex destabilization, nucleic acid aggregation, and rapid protein binding kinetics have been identified as major components of the activity of this highly basic protein (pI ~10). The chaperone activity of other NC proteins is not well understood. We used single molecule DNA stretching to characterize the activity of HIV-1, RSV, and MMLV NC. We found distinct differences in the chaperone activities of each protein, which reflect the requirements for nucleic acid chaperone activity in each retroviral replication system. HTLV-1 NC exhibited overall poor nucleic acid chaperone acitivity. This result is explained by its poor aggregating activity and slow dissociation from single-stranded DNA. This NC protein is overall neutral at pH=7.5 and possesses a unique, acidic C-terminal domain. By studying different HTLV-1 NC mutants, the role of C-terminal domains to the chaperone activity was elucidated. The results suggest that the electrostatic interaction between HTLV-1 NC and nucleic acids is the major factor determining the kinetics. We also examine the nucleic acid interaction properties of the Apolipoprotein B mRNA editing enzyme, a catalytic polypeptide-like 3G (APOBEC3G/A3G) that is known to inhibit HIV-1 reverse transcription in absence of viral infectivity factor (Vif). Our stretching experiments suggested a novel mechanism for deaminase-independent inhibition of reverse transcription due to vital differences of nucleic acid binding kinetics between NC, A3G and reverse transcriptase (RT). Finally, Long interspersed nucleic elements (LINE) are highly repeated nucleic acids sequences in mammal genomes. Our single DNA molecule stretching experiments characterized the nucleic acid chaperone function of ORF1p in the mouse LINE-1 retrotransposon. We found that a single amino acid substitution altered retrotransposition efficiency by a factor of 15 due to a reduction in nucleic acid chaperone activity exhibited by ORF1p. For all of the studies presented here, we used single molecule methods to characterize the nucleic acid interactions of proteins involved in reverse transcription in retroviruses or retrotransposons. In each case, complementary bulk experiments were done by collaborators. The results are presented together in each chapter of the thesis.
Wang, Fei, "Single molecule force spectroscopy studies of DNA binding and chaperone proteins" (2008). Physics Dissertations. Paper 7. http://hdl.handle.net/2047/d10016879
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