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https://hdl.handle.net/2142/35267
Description
Title
Single molecule studies of helicase mechanisms
Author(s)
Stevens, Benjamin Cruser
Issue Date
2006
Department of Study
Physics
Discipline
Physics
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Helicase
helicase mechanisms
RecQ
Duplex DNA
Holliday
G-dquadruplexes
Bloom helicase (BLM)
homogolous
Single molecule fluorescence resonance energy transfer (smFRET)
ATP-depandent
translocation
GQ Structure
bloom syndrom
T10
Poly-ethylene glycol (PEG) slides
growth expression
dyes
dipole orientation
small unilamellar vesicle (SUV)
rotational dynamics
gel phase
polarization
Language
en
Abstract
Bloom helicase (BLM) is a member of the RecQ helicase family, a group of 3’ to 5’ helicases which are homologs of the RecQ protein found in E. coli. Besides the canonical helicase function of unwinding duplex DNA, the RecQ family of helicases is known to preferentially bind to non-canonical DNA structures such as G-quadruplexes and Holliday junctions. Ensemble biochemical research has shown that BLM probably has roles in the maintenance of genome integrity by disrupting non-canonical DNA structures and migrating Holliday junctions to allow for continuation of replication forks and proper homologous recombination. However, higher spatial and time resolutions, as well as the ability to look at molecules one at a time instead of in an ensemble, have been shown to be helpful in elucidating the specific actions in many biological systems. Single molecule FRET (smFRET) microscopy provides these abilities with a distance scale of 2-8 nm, 8 ms time resolution, and the ability to look at single molecules. smFRET experiments on BLM have shown several interesting new behaviors which are unavailable to ensemble experiments, and have also confirmed previously known behaviors. BLM is able to disrupt GQ structures without ATP and even in the presence of K+, which stabilizes the GQ structure. Like some other helicases, BLM exhibits an annealing activity which competes with its canonical helicase (unwinding) activity. Translocation of BLM along a single strand has been directly visualized, and it is suggested that BLM goes in the 5’ to 3’ direction at least part of the time. Additionally, a repetitive motion on single strand DNA was observed, which may indicate that BLM repeatedly shuttles along DNA to disrupt non-canonical DNA structures. This same behavior is observed not only during translocation along a single strand, but also when BLM is unwinding duplex DNA. This repetitive unwinding was shown to be due to a single species rather than different proteins coming in from solution. Additionally, there is an ATP-dependent resetting step that is unrelated to translocation. The HRDC domain, which has an auxiliary DNA binding site, is not required for this repetitive motion to occur.
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