This Program Project reflects a confluence of interests of faculty members at the University of Southern California Main Campus and Medical School. The Program is tightly focused on exploring an important human genomic problem, the biochemical and genetic basis for expansions of trinucleotide repeats associated with neurological diseases. Many of the specific aims offer a novel perspective which we believe is unique to the group of investigators at USC. A significant advantage of this Project is the breadth of expertise of the investigators in studying genetic instability from a variety of different perspectives. The Program Project contains three research projects and three core facilities: a Biochemical-Analytical Core, a Mouse core and an Administrative Core. The goal of Project 1 is to investigate the dynamics of how trinucleotide repeat sequences expand over time, using an experimental in vitro system containing """"""""seed"""""""" repeat sequences and different combinations of replication proteins, and a convenient in vivo system in which large. biologically relevant, repeat expansions can be monitored in wild type and mutant procaryotic cells. Insight into the area of expansion dynamics is crucial to understanding this process at the molecular level. Unique to our approach is a combination of expansion kinetics data, using experimental model systems with """"""""state-of the art"""""""" theoretical analysis involving molecular dynamics calculations. Project I will use the facilities of """"""""Biochemical-Analytical"""""""" Core. The goals of Project 2 are first to examine the role of the enzyme FEN-1 in trinucleotide repeat instability in an animal model. Recently, experiments in yeast carrying large trinucleotide repeat tracts showed that cells defective in the Okazaki fragment processing enzyme FEN-1 had a sizable fraction of expansion mutations compared to wild type cells. Genetic instability at microsatellite loci in mice homozygous for a knockout mutation of FEN-1 will be studied using single molecule analysis of DNA from somatic and germline cells. The second goal of Project 2 is to examine the detailed biochemical properties of FEN-1 and other Okazaki fragment processing enzymes to determine their possible roles in genetic instability. Project 2 will rely heavily on both the Mouse core and the Biochemical-Analytical Core. Project 3 will generate the targeted deletion of the murine FEN-1 gene and/or replacement with an inducible version. The impact of FEN-1 deletion on chromosome stability will be particularly interesting in light of chromosomal changes with age in humans and mice. The impact at the tissue and organismal level of mutant animals will be examined. Finally, the effect of mutations at other loci involved in nonhomologous DNA end joining (NHEJ) will be examined for their effect on genetic instability including triplet repeats. The chromosomal, tissue and organismal effects of these mutations will also be examined in animals as a function of age. Project 3 will require the use of both the Mouse core and the Biochemical-Analytical Core.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Program Projects (P01)
Project #
5P01AG017179-02
Application #
6372398
Study Section
Special Emphasis Panel (ZAG1-PKN-2 (M1))
Program Officer
Mccormick, Anna M
Project Start
2000-04-01
Project End
2003-03-31
Budget Start
2001-04-17
Budget End
2002-03-31
Support Year
2
Fiscal Year
2001
Total Cost
$1,127,595
Indirect Cost
Name
University of Southern California
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Chambers, Eric J; Price, Eric A; Bayramyan, Melina C et al. (2003) Computation of DNA backbone conformations. J Biomol Struct Dyn 21:111-25
Karanjawala, Zarir E; Hsieh, Chih-Lin; Lieber, Michael R (2003) Overexpression of Cu/Zn superoxide dismutase is lethal for mice lacking double-strand break repair. DNA Repair (Amst) 2:285-94
Hartenstine, Michael J; Goodman, Myron F; Petruska, John (2002) Weak strand displacement activity enables human DNA polymerase beta to expand CAG/CTG triplet repeats at strand breaks. J Biol Chem 277:41379-89
Karanjawala, Zarir E; Adachi, Noritaka; Irvine, Ryan A et al. (2002) The embryonic lethality in DNA ligase IV-deficient mice is rescued by deletion of Ku: implications for unifying the heterogeneous phenotypes of NHEJ mutants. DNA Repair (Amst) 1:1017-26
Goodman, Myron F (2002) Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu Rev Biochem 71:17-50
Romero, R M; Rojsitthisak, P; Haworth, I S (2001) DNA interstrand crosslink formation by mechlorethamine at a cytosine-cytosine mismatch pair: kinetics and sequence dependence. Arch Biochem Biophys 386:143-53
Poltoratsky, V; Woo, C J; Tippin, B et al. (2001) Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation. Proc Natl Acad Sci U S A 98:7976-81
Rojsitthisak, P; Romero, R M; Haworth, I S (2001) Extrahelical cytosine bases in DNA duplexes containing d[GCC](n).d[GCC](n) repeats: detection by a mechlorethamine crosslinking reaction. Nucleic Acids Res 29:4716-23
Karanjawala, Z E; Shi, X; Hsieh, C L et al. (2000) The mammalian FEN-1 locus: structure and conserved sequence features. Microb Comp Genomics 5:173-7
Hartenstine, M J; Goodman, M F; Petruska, J (2000) Base stacking and even/odd behavior of hairpin loops in DNA triplet repeat slippage and expansion with DNA polymerase. J Biol Chem 275:18382-90