DNA replication, recombination and repair (DNA-RRR) are fundamental genetic processes carried out by large multi-protein assemblies. Separation of the double helix into single strands is a key step in each of these processes and during the time it is exposed, the single-strand DMA (ssDNA) is organized and protected by a ssDNA-binding protein. The major ssDNA-binding protein in humans is Replication protein A (RPA), a heterotrimer that not only binds and protects ssDNA, but also interacts with a number of other DNA-RRR proteins. RPA's ability to simultaneously bind ssDNA and multiple proteins suggests that it helps assemble and coordinate the activities of DNA-RRR proteins on the DNA substrate. Despite a wealth of biochemical data demonstrating RPA-protein interactions, few have been mapped precisely and high resolution structural data on RPA-complexes are lacking. Our long-term goal is to understand the role of RPA in the assembly and function of the DNA-RRR machinery. The focus in this project period is on the role of RPA in the initiation of DNA replication by simian virus 40 (SV40), which is a well characterized model for eukaryotic replication that has provided many useful insights into the mechanisms of cell transformation and transcription control. SV40 is also of direct interest because it and related viruses appear to play a direct role in some human cancers. The network of RPA, the SV40 protein Tag, and DNA polymerase-alpha/primase (pol-prim) carries out the initial stages of viral DNA replication. Building upon current success in using an NMR-based strategy to characterize interactions between RPA and Tag, Aim 1 proposes to extend these studies to characterize interactions between RPA and pol-prim.
Aim 2 addresses the structural basis for how the activity of RPA and Tag is coordinated at both the atomic and molecular level using NMR and X-ray scattering approaches.

Public Health Relevance

The relevance of this work is that it will address a critical gap in knowledge of how complicated proteins are able to work together to ensure that our DNA is properly replicated as cells grow and repaired when it is damaged. Defects in the proteins that perform these functions can lead to cancer and other diseases. These studies will also contribute knowledge about how a virus replicates its DNA in a host organism, which in turn provides ideas about how the virus might be neutralized.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065484-08
Application #
7760098
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Hagan, Ann A
Project Start
2003-02-01
Project End
2011-12-31
Budget Start
2010-02-01
Budget End
2011-12-31
Support Year
8
Fiscal Year
2010
Total Cost
$329,505
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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Thompson, Matthew K; Ehlinger, Aaron C; Chazin, Walter J (2017) Analysis of Functional Dynamics of Modular Multidomain Proteins by SAXS and NMR. Methods Enzymol 592:49-76
Guilliam, Thomas A; Brissett, Nigel C; Ehlinger, Aaron et al. (2017) Molecular basis for PrimPol recruitment to replication forks by RPA. Nat Commun 8:15222
O'Brien, Elizabeth; Holt, Marilyn E; Thompson, Matthew K et al. (2017) The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport. Science 355:
Holt, Marilyn E; Salay, Lauren E; Chazin, Walter J (2017) A Polymerase With Potential: The Fe-S Cluster in Human DNA Primase. Methods Enzymol 595:361-390
Patrone, James D; Pelz, Nicholas F; Bates, Brittney S et al. (2016) Identification and Optimization of Anthranilic Acid Based Inhibitors of Replication Protein?A. ChemMedChem 11:893-9
Bass, Thomas E; Luzwick, Jessica W; Kavanaugh, Gina et al. (2016) ETAA1 acts at stalled replication forks to maintain genome integrity. Nat Cell Biol 18:1185-1195
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Zhao, Weixing; Vaithiyalingam, Sivaraja; San Filippo, Joseph et al. (2015) Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry. Mol Cell 59:176-87
Ning, Boting; Feldkamp, Michael D; Cortez, David et al. (2015) Simian virus Large T antigen interacts with the N-terminal domain of the 70 kD subunit of Replication Protein A in the same mode as multiple DNA damage response factors. PLoS One 10:e0116093

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