Saccharomyces cerevisiae Pif1, an ATP-dependent superfamily 1B helicase, is conserved from yeast to mammals and has been implicated in both mitochondrial and nuclear functions. Using pre-steady-state kinetic analysis, DNA unwinding activity of Pif1 helicase was characterized in order to understand its mechanism of action. Pif1 unwinding of substrates consisting of varying duplex lengths was fit globally to a model for stepwise unwinding which resulted in a forward rate of 75 bp/s with a kinetic step-size of 1 base pair for this enzyme. The measurement of the burst amplitudes revealed that the active form of Pif1 required for optimal unwinding activity is likely a dimer.
The reported preference of Pif1 for unwinding RNA:DNA heteroduplexes was investigated in order to determine the specific kinetic events that lead to this preferential activity. It was demonstrated that the preferential unwinding of the hybrid duplexes was due to neither specific binding nor differences in the rate of strand separation. Instead, Pif1 helicase unwound RNA:DNA hybrids with moderately greater processivity compared to its duplex DNA:DNA counterparts. This higher processivity of Pif1 was attributed to its slower dissociation from the hybrid structures. Biologically, this preferential role of Pif1 may contribute to its functions at both telomeric and nontelomeric sites.
Another important property of this helicase that was investigated is its ability to exert force and displace proteins bound to nucleic acids. Nucleoprotein complexes may serve as obstacles to various cellular pathways. The ability of Pif1 helicase to remove protein barriers from specific DNA sites, such as G-quadruplexes and telomeres was explored. This analysis demonstrated that Pif1 can catalyze the displacement of a quadruplex-binding protein, Sub1, from DNA secondary structures and this activity of the helicase was proposed to be regulated through its interaction with Sub1. It was also demonstrated that Pif1 dislodges the telomere-capping protein, Cdc13 from an 11mer derived from the yeast telomere sequence. The biochemical mechanism for protein displacement by Pif1 helicase was examined using DNA base substitution approach and the results supported a ‘stepwise’ model. Biologically, this function of Pif1 is implicated in overcoming replication blocks at G-quadruplex and telomeric regions.
|Advisor:||Raney, Kevin D.|
|Commitee:||Davidson, Mari K., Eoff, Robert L., Tackett, Alan J., Varughese, Kottayil I.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 77/11(E), Dissertation Abstracts International|
|Keywords:||1B helicase, DNA unwinding, RNA:DNA hybrids|
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