Dissertation/Thesis Abstract

Structure-function studies of the superfamily 1B helicases Dda and Pif1
by Blair, Lauren Paige, Ph.D., University of Arkansas for Medical Sciences, 2008, 238; 3340138
Abstract (Summary)

Helicases are proteins that unwind double-stranded nucleic acids. Dda helicase from bacteriophage T4 has served as an excellent model for understanding the molecular mechanism of this class of enzymes. Study of the structure of Dda may reveal why some helicases translocate in a 5'-to-3' direction on DNA, while others translocate in a 3'-to-5' direction. Attaining a structure of Dda has proven difficult because the protein fails to readily form crystals and is too large for NMR. Also, Dda has been shown to interact with the T4 single stranded DNA binding protein, gp32. We have developed a homology model of the enzyme that we are using to guide further studies that will examine the structural and functional significance of the interaction between Dda and gp32, and how this interaction impacts translocation on DNA. We tested the structural model by examining the protein surface using two common methods for mapping protein domains and for examining protein surfaces: limited proteolysis and chemical footprinting. We also performed a functional analysis of Dda variants with amino acid alterations at sites known to interact with DNA in other helicases. In the process, we identified the site of Dda interaction on DNA as well as the site of Dda interaction with gp32. The Pif1 helicase from Saccharomyces cerevisiae is in the same superfamily and has the same directionality as Dda. We identified the interactome of Pif1 and found that it interacts with Rim1, a mitochondrial SSB. We also performed a basic analysis of Pif1 post translational modifications. These studies taken together allow us to present a mechanism for SF1B helicases taking into account their interaction with DNA and SSBs.

Indexing (document details)
Advisor: Raney, Kevin D.
Commitee:
School: University of Arkansas for Medical Sciences
School Location: United States -- Arkansas
Source: DAI-B 69/12, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology, Biochemistry
Keywords: Dda, Helicases, Mass spectrometry, Pif1
Publication Number: 3340138
ISBN: 978-0-549-94381-5
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