Replication of DNA is a complex multi-step process essential to cell propagation and survival, which proceeds via the action of multi-protein machines. Understanding the machinery at the replication fork has high impact because it is the most critical site for propagation and maintenance of the genome. While considerable progress has been made in elucidating the mechanisms of DNA replication from studies of bacteria and archae, information on replication in humans is lacking because the protein sequences and structures are not conserved. The long-term goal of our research is to understand the action of the DNA replication machinery in humans. Our research currently focuses on understanding the initiation of the step known as DNA priming. We have shown active loading of human replication protein A (RPA) onto single-stranded DNA (ssDNA) created by the SV40 helicase at the origin of replication and involvement of RPA in the transition to DNA priming. After the DNA is unwound, an initial primer is synthesized on the ssDNA template by primase. The studies proposed here are designed to generate insight into how RPA and primase function together to initiate synthesis of the primer strand.
Aim 1 investigates the structure of RPA in different DNA-bound states using a combination of small angle X-ray and neutron scattering (SAXS, SANS) and NMR spectroscopy.
Aim 2 addresses the role of interactions with RPA in promoting the loading of primase on the template using a combination of biochemical mapping and structural analyses by NMR, modeling, SAXS and SANS. Once primase is loaded on the DNA template, it synthesizes a ~10 nucleotide primer and then transfers the primed template to DNA polymerase a for primer extension. The means by which primase recognizes the template and counts the length of the primer remains a complete mystery.
Aim 3 proposes to elucidate the structural basis for these processes by determining x-ray crystal structures of primase in different DNA bound states. Together, these results will inform the structural basis for the hand-off of ssDNA from RPA to DNA primase and counting of the RNA primer, which are critical steps in the replication of DNA.

Public Health Relevance

Faithful replication and maintenance of our genomes requires the action of complex multi-protein machinery. Defects in components of this machinery lead to mutation and ultimately cancer and other diseases associated with genomic instabilities. Investigating the structure and coordinated action of the complex of proteins involved in initiating replication in humans will provide detailed mechanistic insight into the action of the multi-protein machinery at the replication fork, the most critical site for propagation and maintenance of the genome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065484-10
Application #
8403077
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Reddy, Michael K
Project Start
2003-02-01
Project End
2015-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
10
Fiscal Year
2013
Total Cost
$324,414
Indirect Cost
$116,456
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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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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Chen, Jin; Le, Shimin; Basu, Anindita et al. (2015) Mechanochemical regulations of RPA's binding to ssDNA. Sci Rep 5:9296
Sugitani, Norie; Chazin, Walter J (2015) Characteristics and concepts of dynamic hub proteins in DNA processing machinery from studies of RPA. Prog Biophys Mol Biol 117:206-11
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
Frank, Andreas O; Vangamudi, Bhavatarini; Feldkamp, Michael D et al. (2014) Discovery of a potent stapled helix peptide that binds to the 70N domain of replication protein A. J Med Chem 57:2455-61
Feldkamp, Michael D; Mason, Aaron C; Eichman, Brandt F et al. (2014) Structural analysis of replication protein A recruitment of the DNA damage response protein SMARCAL1. Biochemistry 53:3052-61
Vaithiyalingam, Sivaraja; Arnett, Diana R; Aggarwal, Amit et al. (2014) Insights into eukaryotic primer synthesis from structures of the p48 subunit of human DNA primase. J Mol Biol 426:558-69

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