Complete and faithful duplication of the genome is a fundamental prerequisite for cell division in development and tissue renewal. The protein machinery that replicates DNA is part of a highly integrated protein network that maintains genomic stability in all cells. Errors and malfunctions in genome maintenance can result in loss of cell viability and are responsible in large part for diseases such as cancer, including virus-associated cancers. However, major gaps remain in our knowledge of how the mammalian genome is duplicated, how this process is regulated, and how malfunctions are corrected. The long-term goal of the proposed research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. Chromosomal DNA replication begins with the synthesis of RNA primers that can be extended by a DNA polymerase. In prokaryotes, a dynamic nanomachine known as a 'primosome'couples duplex DNA unwinding with RNA primer synthesis, but eukaryotic primosomes remain elusive. Analysis of simian virus 40 (SV40) DNA replication provides the first insight into a eukaryotic primosome that can be reconstituted with purified recombinant proteins in vitro. The SV40 primosome consists of the viral helicase large T antigen (Tag), the host DNA polymerase alpha-primase (pol-prim), and the host single-strand DNA-binding protein RPA. Our previous work suggests that a network of at least six pairs of physical interactions of the viral helicase Tag with human pol-prim and RPA gives rise to primosome activity. Similarly, a conserved vertebrate helicase B (HELB/HDHB) interacts with pol-prim and RPA, and displays primosome activity in vitro. HDHB has been implicated in chromosomal replication and in replication restart after DNA damage in human cells. The proposed research program is designed (1) to develop a comprehensive mechanistic understanding of the SV40 primosome at the atomic level, and (2) to extend this mechanistic analysis to the HDHB primosome activity and determine whether this activity is linked to HDHB-mediated replication restart after DNA damage. We anticipate that these "simple" primosome mechanisms will serve as useful models for identifying and understanding the complex primosome(s) that initiates chromosomal replication in eukaryotes and others that may restart replication forks arrested by DNA damage.

Public Health Relevance

Accurate and timely replication of genomic DNA is a fundamental prerequisite for the propagation of each cell in all kingdoms of life. Cells have evolved a complex network of proteins to maintain genome stability in the face of environmental and endogenous DNA damage. Since viruses have evolved to exploit these pathways for their own propagation, often with pathogenic consequences for the host, e.g. cancer, discovery of these pathways will uncover new targets for the design of anti-viral drugs and more effective cancer therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052948-18
Application #
8462987
Study Section
Virology - A Study Section (VIRA)
Program Officer
Reddy, Michael K
Project Start
1995-08-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
18
Fiscal Year
2013
Total Cost
$491,500
Indirect Cost
$176,436
Name
Vanderbilt University Medical Center
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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
Sowd, Gregory A; Li, Nancy Yan; Fanning, Ellen (2013) ATM and ATR activities maintain replication fork integrity during SV40 chromatin replication. PLoS Pathog 9:e1003283
Guler, Gulfem Dilek; Liu, Hanjian; Vaithiyalingam, Sivaraja et al. (2012) Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress. J Biol Chem 287:6469-81
Huang, Hao; Zhao, Kun; Arnett, Diana R et al. (2010) A specific docking site for DNA polymerase {alpha}-primase on the SV40 helicase is required for viral primosome activity, but helicase activity is dispensable. J Biol Chem 285:33475-84
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Wright, Christine M; Seguin, Sandlin P; Fewell, Sheara W et al. (2009) Inhibition of Simian Virus 40 replication by targeting the molecular chaperone function and ATPase activity of T antigen. Virus Res 141:71-80
Warren, Eric M; Huang, Hao; Fanning, Ellen et al. (2009) Physical interactions between Mcm10, DNA, and DNA polymerase alpha. J Biol Chem 284:24662-72
Fanning, Ellen; Zhao, Kun (2009) SV40 DNA replication: from the A gene to a nanomachine. Virology 384:352-9

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