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.
|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|
|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|
|Rad, Behzad; Kowalczykowski, Stephen C (2012) Translocation of E. coli RecQ helicase on single-stranded DNA. Biochemistry 51:2921-9|
|Carreira, Aura; Kowalczykowski, Stephen C (2011) Two classes of BRC repeats in BRCA2 promote RAD51 nucleoprotein filament function by distinct mechanisms. Proc Natl Acad Sci U S A 108:10448-53|
|Forget, Anthony L; Kowalczykowski, Stephen C (2010) Single-molecule imaging brings Rad51 nucleoprotein filaments into focus. Trends Cell Biol 20:269-76|
|Hilario, Jovencio; Kowalczykowski, Stephen C (2010) Visualizing protein-DNA interactions at the single-molecule level. Curr Opin Chem Biol 14:15-22|
|Cejka, Petr; Cannavo, Elda; Polaczek, Piotr et al. (2010) DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2. Nature 467:112-6|
|Cejka, Petr; Plank, Jody L; Bachrati, Csanad Z et al. (2010) Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3. Nat Struct Mol Biol 17:1377-82|
Showing the most recent 10 out of 57 publications