Abstract

The studies described in this research proposal are directed at determining the biochemical mechanism and biological function of DNA helicases. The proteins to be investigated include the E. coli recBCD enzyme, rep protein, and uvrD protein. These proteins play important roles in the processes of DNA replication, recombination and DNA repair. The biochemical mechanism of helicase action will be investigated by taking advantage of a rapid, continuous, and accurate fluorescence helicase assay for DNA unwinding that has been recently developed in my laboratory. This assay poses several advantages over traditional methods and will be used to biochemically characterize the helicase activities of the recBCD enzyme, rep protein and uvrD protein. In addition, the relationship of the ATPase activity of these helicases to their DNA unwinding activity will be examined. To better understand the physical basis of the DNA unwinding activity, the equilibrium DNA and ATP binding properties of these protein will be examined. All of these studies will also be extended to a characterization of the biochemical properties of mutant DNA helicases in order to attempt to relate the biochemical activities with biological function.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041347-02
Application #
3299482
Study Section
Biochemistry Study Section (BIO)
Project Start
1988-12-01
Project End
1993-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
2
Fiscal Year
1990
Total Cost
Indirect Cost

  Institution

Name
Northwestern University at Chicago
Department
Type
Schools of Dentistry
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611

  Publications

Bell, Jason C; Kowalczykowski, Stephen C (2016) RecA: Regulation and Mechanism of a Molecular Search Engine. Trends Biochem Sci 41:491-507
Pavankumar, T L; Exell, J C; Kowalczykowski, S C (2016) Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases. Methods Enzymol 581:1-32
Fasching, Clare L; Cejka, Petr; Kowalczykowski, Stephen C et al. (2015) Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism. Mol Cell 57:595-606
Kowalczykowski, Stephen C (2015) An Overview of the Molecular Mechanisms of Recombinational DNA Repair. Cold Spring Harb Perspect Biol 7:
Bocquet, Nicolas; Bizard, Anna H; Abdulrahman, Wassim et al. (2014) Structural and mechanistic insight into Holliday-junction dissolution by topoisomerase III? and RMI1. Nat Struct Mol Biol 21:261-8
Paeschke, Katrin; Bochman, Matthew L; Garcia, P Daniela et al. (2013) Pif1 family helicases suppress genome instability at G-quadruplex motifs. Nature 497:458-62
Liu, Bian; Baskin, Ronald J; Kowalczykowski, Stephen C (2013) DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate. Nature 500:482-5
Handa, Naofumi; Yang, Liang; Dillingham, Mark S et al. (2012) Molecular determinants responsible for recognition of the single-stranded DNA regulatory sequence, ?, by RecBCD enzyme. Proc Natl Acad Sci U S A 109:8901-6
Cejka, Petr; Plank, Jody L; Dombrowski, Christopher C et al. (2012) Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA complex: a mechanism for disentangling chromosomes. Mol Cell 47:886-96
Rad, Behzad; Kowalczykowski, Stephen C (2012) Efficient coupling of ATP hydrolysis to translocation by RecQ helicase. Proc Natl Acad Sci U S A 109:1443-8

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