The long-term goal of our research is to understand the molecular mechanisms by which the eukaryotic single-stranded DNA-binding protein, Replication Protein A (RPA), interacts with single-stranded-DNA (ssDNA) and with other proteins in various DNA-processing events. An interaction of RPA with ssDNA is considered to a sequence non-specific. Surprisingly, structural analysis demonstrated that the RPA forms multiple specific hydrogen-bond contacts to cytidine bases in the crystal structure.
The aims of the studies described in this proposal are to complete the structural analysis of the remaining ssDNA-binding domains to investigate the DNA-binding induced conformational changes in ssDNA-binding domain and understand a mechanism of sequence non- specificity. To achieve our goals, we will use a combination of biochemical methods and X-ray crystallographic analysis. Specifically our aims are: 1. Does ssDNA binding induce a conformational change in the two ssDNA-binding domains in the largest RPA subunit (RPA70)? The crystal structure of the two ss-DNA binding domains in RPA70 (RPA70/181-422 fragment) will be determined in the absence of ssDNA. 2. What structural features allow the interaction of the two RPA70 ssDNA binding domains with ssDNA to be sequence non-specific? Structures of RPA70181/422 in complex with (dT)8, (dG)8 and (dA)8 will be crystallized and determined to analyze an interaction with each type of DNA base in each position. 3. Is the C-terminal domain of the largest RPA subunit (RPA70) structurally homologues to other ssDNA-binding domains of RPA? What is the mechanism of the RPA trimerisation?. A structure of the C- terminal domain of RPA70 in complex with two smaller subunits, RPA32 and RPA14, will be resolved by X-ray crystallography. 4. What is the general mechanism of ssDNA-binding by the RPA trimer? A trimeric RPA complex containing all ssDNA-binding domains will be resolved by X-ray crystallography with and/or without ssDNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061192-03
Application #
6520228
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
2000-03-01
Project End
2005-02-28
Budget Start
2002-03-01
Budget End
2003-02-28
Support Year
3
Fiscal Year
2002
Total Cost
$181,875
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
937727907
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
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Bochkarev, Alexey; Bochkareva, Elena (2004) From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold. Curr Opin Struct Biol 14:36-42
Pestryakov, Pavel E; Khlimankov, Denis Y; Bochkareva, Elena et al. (2004) Human replication protein A (RPA) binds a primer-template junction in the absence of its major ssDNA-binding domains. Nucleic Acids Res 32:1894-903
Yoo, Byong Hoon; Bochkareva, Elena; Bochkarev, Alexey et al. (2004) 2'-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro. Nucleic Acids Res 32:2008-16
Arunkumar, Alphonse I; Stauffer, Melissa E; Bochkareva, Elena et al. (2003) Independent and coordinated functions of replication protein A tandem high affinity single-stranded DNA binding domains. J Biol Chem 278:41077-82
Bochkareva, Elena; Korolev, Sergey; Lees-Miller, Susan P et al. (2002) Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA. EMBO J 21:1855-63
Bochkareva, E; Belegu, V; Korolev, S et al. (2001) Structure of the major single-stranded DNA-binding domain of replication protein A suggests a dynamic mechanism for DNA binding. EMBO J 20:612-8
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