Project Description: The long-term objective of this proposal is to understand both the biochemical mechanism and biological function of DNA helicase action: i.e., how helicases translocate along single-stranded DNA (ssDNA) to unwind double-stranded DNA (dsDNA) and, how this effort is translated into biological function. Furthermore, one of the helicases (RecBCD) that is a major focus of this proposal is distinctive in that it recognizes a specific sequence (?) while translocating and, in response, alters its biochemical behavior. The second part of this project deals with the RecQ-family of helicases, which is ubiquitous and has well-defined members in bacteria, yeasts, and humans. The RecQ helicases are seemingly less complex, but they function in many aspects of DNA metabolism and their complexity stems from the fact that they work in conjunction with partner proteins to effect functionally important changes in DNA structure. This grant proposal has 2 broad specific aims. The first is to study RecBCD enzyme using molecular, biochemical, and single-molecule approaches to determine how recognition of a ? sequence reversibly switches both the structure and function of RecBCD enzyme, and also to establish when and where the ?- activated enzyme binds RecA protein, and how it is loaded onto the ?-containing single-stranded DNA.
The second aim i s to visualize DNA unwinding by the RecQ helicases, determine how the unwinding behavior is altered by interaction with partner proteins, and determine when and where the partner proteins associate with the helicase. Understanding the mechanism and function of these motor proteins is a longstanding goal of this research proposal.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041347-24
Application #
8450108
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Barski, Oleg
Project Start
1988-12-01
Project End
2016-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
24
Fiscal Year
2013
Total Cost
$473,980
Indirect Cost
$148,509
Name
University of California Davis
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Bell, Jason C; Kowalczykowski, Stephen C (2016) RecA: Regulation and Mechanism of a Molecular Search Engine. Trends Biochem Sci 41:491-507
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
Bocquet, Nicolas; Bizard, Anna H; Abdulrahman, Wassim et al. (2014) Structural and mechanistic insight into Holliday-junction dissolution by topoisomerase IIIα 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, χ, 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
Yang, Liang; Handa, Naofumi; Liu, Bian et al. (2012) Alteration of χ recognition by RecBCD reveals a regulated molecular latch and suggests a channel-bypass mechanism for biological control. Proc Natl Acad Sci U S A 109:8907-12
Rad, Behzad; Kowalczykowski, Stephen C (2012) Translocation of E. coli RecQ helicase on single-stranded DNA. Biochemistry 51:2921-9

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