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.
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