Pif1-family helicases play various roles in the maintenance of nuclear and mitochondrial genome integrity in most eukaryotes. Saccharomyces cerevisiae Pif1 helicase is the prototypical member of the Pif1-family of DNA helicases. Pif1 helicase was characterized using presteady-state kinetics to determine rates of DNA unwinding, translocation, and ATP hydrolysis. Pif1 unwinds dsDNA with a rate of ~75 bp/s, and with kinetic step-size of 1 bp. The rate of translocation on ssDNA (~89 nt/s) is essentially the same as the rate of unwinding of dsDNA unwinding; therefore Pif1 is an active helicase. The ATPase activity of Pif1 during translocation was 0.84 ATP/nucleotides translocated. Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of one molecule of ATP. Pif1's ability to displace streptavidin from biotinylated oligonucleotides indicates that it can generate force to displace proteins. The mechanism of telomere regulation by Pif1 could be mediated through its protein displacement activity to displace the telomerase complex from DNA. Pif1's role in multiple biological functions suggested that it may have interacting partners. To characterize the Pif1 interactome, a proteomics technique called I-DIRT was employed. I-DIRT helps to differentiate specific protein-protein interactions from non-specific. A stable association between Pif1 and the mitochondrial single-stranded binding protein, Rim1, was identified. The observed dissociation constant for the Pif1-Rim1 interaction was 0.69 µM. The functional significance of the Pif1-Rim1 interaction was tested by studying Pif1 helicase activity in the presence or absence of Rim1. Pif1 DNA helicase activity was stimulated by 4- to 5-fold in the presence of Rim1. A coordinated interaction between Pif1 and Rim1 could play a role in mitochondrial DNA metabolism. Biochemical analysis to test Pif1-catalyzed strand-annealing led to discovery of a new activity for this helicase. Pif1 showed robust strand-annealing activity in the presence and absence of ATP. However, longer duplexes and 3'-overhang substrates acted as the best substrates for annealing in the presence of ATP because these were poor substrates for unwinding activity due to the 5'→3' directionality. Pif1 efficiently annealed strands with mismatches in their complementary sequence, indicating that it can function even when strands are not fully complementary. Although biological significance of Pif1 strand-annealing activity need to determined, the biochemical analysis and previous reports indicate that Pif1 annealing activity could be used in the double-strand break (DSB) repair pathways. It is possible that a coordinated action of unwinding and annealing activities may be required to achieve all of the reported biological functions of Pif1.
|Advisor:||Raney, Kevin D.|
|Commitee:||Baldini, Giulia, Kottayil, Varughese, Miller, G. Paul, Tackett, Alan J.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 75/05(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry|
|Keywords:||Dna helicase, Dna unwinding, Pif1 helicase, Protein displacement, Protein-protein interaction, Strand annealing|
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