This competing renewal in its 30th year continues a strong record of studies of DNA replication, in particular the architecture of moving forks. The unique approach couples electron microscopic (EM) examination of DNA- protein complexes, biochemical analysis, and the use of genetic mutants. Previous work supported by this grant provided the first direct proof that the lagging strand at a fork loops back (trombone model) and that there are 2 (and sometimes 3) polymerase molecules present. DNA replication in human and animal mitochondria (mt) is central to aging and DNA damage. Recent work by Jacobs and others has revealed that human mtDNA may replicate via modes not previously thought to occur, including the generation of recombinational networks. In deciphering these mechanisms, the EM approaches employed will be critical in establishing the architecture of the replication intermediates. These studies will continue as collaborations with Howard Jacobs and Laurie Kaguni's labs and will focus on the Drosophila mtDNA system where powerful genetic tools are available. Comparative studies of Drosophila and human mt. single strand (ss) binding proteins and their functional variants will be examined in their binding to ssDNA. In addition to examining purified mtDNA, mtDNA-protein complexes from human and Drosophila cells will be examined by EM. Work in progress on the novel rolling circle replication of nematode mtDNA will be continued. In order to understand basic modes of replication in human cells, animal viruses are powerful tools and the Herpes Simplex virus type 1 has provided an excellent model system due to the simple set of 6 replication factors used and that three modes of replication: rolling circle theta, and recombination-coupled replication may be employed in vivo. All 6 HSV-1 replication proteins have been highly purified in this laboratory and using them, a recent groundbreaking advance has been the reconstitution in vitro of robust rolling circle replication which generates tails of >25 kb from circular ?X174 templates. At later incubation times the reactions generate recombinational DNA networks, providing the first model system for studying this poorly understood replication mode. These advances set the stage for rapid progress bolstered by collaboration with Sandra Weller's group who will provide a large number of mutants in the proteins. The studies will focus on understanding the mechanism of all three replication modes and whether there is a lagging strand loop. The number of polymerase and helicase/primase molecules at a moving fork will be determined using a tool developed in the last renewal, nano-scale bipointers. A new EM method (cryo-shadowing) will be applied to achieve better structural resolution. These experiments will test our hypothesis that looping of the lagging strand and the general features of the fork architecture are broadly shared across widely different systems.

Public Health Relevance

. All living organisms must replicate their DNA. These studies focus on mechanisms that control DNA replication in mitochondria and human cells, to provide the basis for anti-cancer therapies, drugs to target microbial infections, and a more refined understanding of the aging process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM031819-29
Application #
8692194
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Reddy, Michael K
Project Start
1983-04-01
Project End
2018-03-31
Budget Start
2014-04-11
Budget End
2015-03-31
Support Year
29
Fiscal Year
2014
Total Cost
$358,758
Indirect Cost
$122,733
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
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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
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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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