Helicases are ubiquitous molecular motors that couple the energy released during nucleotide tri-phosphate hydrolysis to unwind and translocate nucleic acids. Helicases provide the single-stranded intermediates necessary for virtually all aspects of nucleic acid metabolism making them desirable targets for mechanistic studies. The mechanism of DNA unwinding by helicases has been extensively studied in recent years but is still not completely understood. I investigated several aspects of the mechanism of DNA unwinding by the bacteriophage T4 Dda helicase. The interaction of helicases with nucleic acid substrates that the helicase might encounter in vivo has been observed through several studies including DNA footprinting, enzyme kinetics, site-directed mutagenesis, and application of chemically modified nucleic acid substrates.
In the case of Dda helicase, the translocation rate on ssDNA and the rate of unwinding of duplex DNA were found to be similar suggesting that Dda is a perfectly active helicase. One of the least understood aspects of helicase-catalyzed duplex DNA unwinding is the role of protein interactions with the displaced strand. The active nature of Dda could be explained in part on the basis of its interaction with the translocating as well as displaced strand during duplex DNA unwinding.
By employing pre-steady state DNA unwinding and DNA footprinting approaches, I observed that interactions between the displaced strand and Dda occur during unwinding of a fork DNA. In the presence of saturating Dda, unwinding of a fork DNA resulted in an increased product formation as compared to a ss/dsDNA junction substrate. The lag phase of the reaction progress curve was reduced during the unwinding of forked DNA. Consequently, fewer steps were required to unwind the fork DNA. The crystal structure of Dda reported a group of basic residues within a region of the 1A domain, which could be a potential site for binding to the displaced strand. Replacement of basic residues (K24, K25) located on this site with alanines reduced the ability of Dda to interact with the displaced strand. Neutralization of the backbone charges of the displaced strand of DNA using methyl phosphonate modifications disrupted the binding interaction of Dda with the displaced strand. Taken together, these results suggested that multiple interactions of Dda with the tracking and displaced strands coordinate DNA unwinding. This data led to the suggestion of a new model for the Dda-catalyzed helicase mechanism: the directed exclusion of the displaced strand, which contributes to making Dda a perfectly active helicase.
|Advisor:||Raney, Kevin D.|
|Commitee:||Diekman, Alan, Miller, Paul, Tackett, Alan, Varughese, Kottayil|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 74/06(E), Dissertation Abstracts International|
|Keywords:||Active helicase, Bacteriophage T4 Dda helicase, DNA unwinding, Translocation|
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