Effects of Site-directed Mutagenesis of Backbone Tracking Residues on the Behavior of Rep Helicase Translocation
Helicases are essential motor proteins that unwind the double helix molecule of DNA to yield single strand DNA (ssDNA) intermediates required for replication, recombination and repair. These enzymes unwind duplex DNA and translocate from the 3' to 5 ' end along DNA in reactions coupled to the binding and hydrolysis of 5' nucleotide triphosphates. By inducing site-directed mutagenesis on backbone tracking residues Threonine 83 and 556 in the super family 1 (SF1) helicase protein, Rep, in Escherichia coli, we hoped to elucidate if the translocation behavior of this helicase could be altered. Modifying these residues in Rep would help to understanding the mechanism that helicases use to translocate and unwind DNA and thus facilitate better understanding of more complex molecular motors. Molecular biology and single-molecule biophysics techniques, such as site-directed mutagenesis, protein expression and purification, and Total Internal Reflection Fluorescence Microscopy (TIRFM) were used to observe individual molecules and verify altered activity of the mutated Rep protein, were performed. Single molecule experiments showed that the translocation of the T556A mutated Rep is four times slower than the control Rep suggesting that there is a slow or ATP-independent hydrolysis coupled to the translocation.
School:
University of Puerto Rico at Humacao
Department:
Biological Sciences / Genetics
Research Advisor:
Taekjip Ha
Department of Research Advisor:
Physics
Year of Publication:
2008