Abstract

Single molecule electron microscopy provides a powerful approach to study the way in which damaged DNA is remodeled by proteins. The focus of this application is understanding how a number of central human DNA repair and telomere binding proteins interact at large, complex DNA structures containing damage, and how they carry out repair or signal the presence of lesions. This is a highly interactive program which represents longstanding fruitful collaborations with Dr. Paul Modrich working on human mismatch factors, Dr. Aziz Sancar working on human repair signaling factors, and with Dr. Titia deLange working on telomere binding proteins. Together from our own work on this topic and through these collaborations we have published over 20 papers in the past 5 years. This is a highly propitious time to carry out these studies since we have developed two powerful new EM methods: nano-scale biopointers that provide a means of identifying the location of proteins within multi-protein complexes and glycerol spray/low voltage EM that gives a more gentle means of preparing samples for EM. Further, as substrates for these studies, we have produced large natural DNAs containing replication forks or Holliday junctions with nearby mismatched bases and a model telomere DNA. Work on the mismatch repair proteins will take advantage of the recent in vitro reconstitution of nick directed excision repair by the Modrich laboratory. Work on Claspin and the Rad 9- Husl-Radl complex will focus on learning how these proteins interact with replication forks containing damage. Studies of the remodeling of telomeres will take advantage of the recent discovery of discrete multi protein complexes at telomeres. Finally continuing work from our laboratory will focus on p53 as a facilitator of DNA damage recognition. Each system offers a unique window into basic questions of DNA protein remodeling at sites of damage and telomeres and information garnered from one study is immediately applied to the others. ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES013773-02
Application #
7101948
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Reinlib, Leslie J
Project Start
2005-07-26
Project End
2010-04-30
Budget Start
2006-05-01
Budget End
2007-04-30
Support Year
2
Fiscal Year
2006
Total Cost
$338,602
Indirect Cost

  Institution

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

  Publications

Nicholls, Thomas J; Nadalutti, Cristina A; Motori, Elisa et al. (2018) Topoisomerase 3? Is Required for Decatenation and Segregation of Human mtDNA. Mol Cell 69:9-23.e6
Kar, Anirban; Jones, Nathan; Arat, N Özlem et al. (2018) Long repeating (TTAGGG) n single-stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation. J Biol Chem 293:9473-9485
Amunugama, Ravindra; Willcox, Smaranda; Wu, R Alex et al. (2018) Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading. Cell Rep 23:3419-3428
Zhu, Cheng; Beck, Matthew V; Griffith, Jack D et al. (2018) Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A 115:4661-4665
Prasad, Rajendra; Ça?layan, Melike; Dai, Da-Peng et al. (2017) DNA polymerase ?: A missing link of the base excision repair machinery in mammalian mitochondria. DNA Repair (Amst) 60:77-88
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
Simonicova, Lucia; Dudekova, Henrieta; Ferenc, Jaroslav et al. (2015) Saccharomyces cerevisiae as a model for the study of extranuclear functions of mammalian telomerase. Curr Genet 61:517-27
Ciesielski, Grzegorz L; Bermek, Oya; Rosado-Ruiz, Fernando A et al. (2015) Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase ? by Organization of the Template DNA. J Biol Chem 290:28697-707
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
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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