Analysis of single molecules by EM provides a powerful approach to study the mechanics of DNA replication. The long-term focus of this study combines biochemistry, protein tagging, with direct visualization of DNA protein complexes by electron microscopy (EM). A statistically large number of molecules are examined and this information is combined with data from biochemical assays. This project has great strength in its long standing interactions with Dr. Steve Bell (yeast ORC), Drs. Tom Broker and Louise Chow (human papillomaviruses (HPV)), and Dr. Charles Richardson (T7 replication). These studies are augmented with work carried out in this laboratory on T4 and Herpes Simplex type 1 (HSV-1). The goal is to understand origin activation (HPV, yeast) and the architecture of a moving fork (T7, T4, HSV-1). A new technology using nano-scale paramagnetic particles attached to specifically designed DNA templates allows visualization of nanogram quantities of DNA and protein and the rapid exchange DNA-protein complexes into buffers optimal for high resolution EM. DNAs containing the yeast ARS1 element attached to magnetic particles will be incubated with yeast cell extracts to load the pre-RC complex including Mcm and Cdc6 factors. Their structure will be probed by Western analysis and EM using nanoscale 'biopointers'. DNA fragments containing the HPV origin bound to paramagnetic particles will be assembled with the E1, E2 proteins as well as factors from 293 cell extracts. The stepwise opening of the origin and its interaction with cell factors including p53 will be investigated using a combination of EM, Western analysis, and replication assays. Continued studies of the moving fork in T7 and T4 will employ paramagnetic particles along with single particle image reconstructions and cryoEM to generate high resolution structures containing the polymerase, helicase-primase, SSBs and other factors on model replication forks. Using HSV-1 replication proteins purified in this laboratory, the architecture of DNA-protein complexes which catalyze replication will be examined to provide information on the looping of the lagging strand in a moving eukaryotic fork. Using these in vitro T4 and HSV-1 replication systems, the way in which recombination coupled replication is initiated by recombination factors and how the replication forks proceed to generate networks of replicating DNA will be visualized. Understanding the structure of the proteins and protein complexes which catalyze replication of prokaryotic and eukaryotic chromosomes and viruses is crucial to the development of drugs which bind key components at replication forks. This will help lead to development of new anti-bacterial, fungal, and viral compounds.

Public Health Relevance

Understanding the structure of the proteins and protein complexes which catalyze replication of prokaryotic and eukaryotic chromosomes and viruses is crucial to the rational design of new drugs which bind these proteins and structures. This study employs high resolution electron microscopy to provide a unique and direct visualization of protein complexes at replication origins and replication forks in bacteria, fungi, eukaryotic cells and viruses to provide the information needed to identify new drug targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031819-27
Application #
7999263
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Janes, Daniel E
Project Start
1983-04-01
Project End
2012-12-31
Budget Start
2011-01-01
Budget End
2011-12-31
Support Year
27
Fiscal Year
2011
Total Cost
$332,683
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Sepsiova, Regina; Necasova, Ivona; Willcox, Smaranda et al. (2016) Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins. PLoS One 11:e0154225
Ciesielski, Grzegorz L; Bermek, Oya; Rosado-Ruiz, Fernando A et al. (2015) Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA. J Biol Chem 290:28697-707
Lewis, Samantha C; Joers, Priit; Willcox, Smaranda et al. (2015) A rolling circle replication mechanism produces multimeric lariats of mitochondrial DNA in Caenorhabditis elegans. PLoS Genet 11:e1004985
Bermek, Oya; Willcox, Smaranda; Griffith, Jack D (2015) DNA replication catalyzed by herpes simplex virus type 1 proteins reveals trombone loops at the fork. J Biol Chem 290:2539-45
Dillon, Laura W; Kumar, Pankaj; Shibata, Yoshiyuki et al. (2015) Production of Extrachromosomal MicroDNAs Is Linked to Mismatch Repair Pathways and Transcriptional Activity. Cell Rep 11:1749-59
Bakkaiova, Jana; Marini, Victoria; Willcox, Smaranda et al. (2015) Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids. Biosci Rep 36:e00288
Holmes, J Bradley; Akman, Gokhan; Wood, Stuart R et al. (2015) Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication. Proc Natl Acad Sci U S A 112:9334-9
Ozgur, Sezgin; Griffith, Jack (2014) Interaction of Kaposi's sarcoma-associated herpesvirus ORF6 protein with single-stranded DNA. J Virol 88:8687-95
Bower, Brian D; Griffith, Jack D (2014) TRF1 and TRF2 differentially modulate Rad51-mediated telomeric and nontelomeric displacement loop formation in vitro. Biochemistry 53:5485-95
Chen, Stefanie Hartman; Plank, Jody L; Willcox, Smaranda et al. (2014) Top3α is required during the convergent migration step of double Holliday junction dissolution. PLoS One 9:e83582

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