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B.Tech, Anna University - 2008
Thesis Advisor: Smita S. Patel
Graduate Program in Biochemistry
School of Public Health Building
2nd floor Conference Room 258
Monday, February 23, 2015
DNA replication is catalyzed by two molecular motors; the replicative DNA helicase and DNA polymerase (DNAP), which use the energy from nucleotide triphosphate hydrolysis to move on DNA. The isolated enzymes translocate efficiently on single-stranded (ss) DNA, but slow down considerably when they encounter double-stranded (ds) DNA that needs to be unwound. However, the combined enzymes have the ability to catalyze rapid and efficient leading strand replication, which requires dsDNA unwinding. Current models assume that helicase unwinds the dsDNA to create ssDNA template for the DNAP that copies the ssDNA to drive DNA unwinding. How DNAP increases the unwinding activity of the helicase is not known. Further, DNAPs have limited unwinding activity on their own, whose role is poorly understood. My thesis research addresses these gaps in the fundamental mechanism of coupling between replicative helicase and DNAP; first, by defining the precise positions of the two enzymes at the replication fork junction, and second, by identifying the kinetic mechanism of functional interdependence using the T7 bacteriophage enzymes as a model system.
Using pre-steady state kinetics, I show that the unwinding rates of T7 helicase or T7 DNAP in isolation are GC-dependent and slower than the translocation rate on ssDNA templates. Increasing dNTP concentration restores fast rates of DNAP, but not those of the helicase whose unwinding rates remain low even at high dTTP (helicase substrate) concentrations. Increasing resistance from dsDNA unwinding lowers the kcat of DNA unwinding for the helicase and increases the dNTPs K¬m for the DNAP, providing evidence for fundamentally different mechanisms of coupling substrate dNTP binding to translocation which is defined by kinetic modeling. In contrast, the coupled helicase-DNAP is not rate-limited by dsDNA unwinding because the DNAP increases the unwinding kcat of the helicase and the helicase decreases the dNTPs Km of the DNAP. Thus, within the coupled system, each enzyme behaves like a motor that is translocating on ssDNA.
To understand the structural basis for the mutual dependence, I used 2-aminopurine fluorescent base as a probe for monitoring DNA unwinding at single base-pair resolution. The study shows that the isolated DNAP or helicase has the ability to melt the junction base pair, but not as efficiently as the combined enzymes that melt the junction base pair cooperatively. Interestingly, this cooperativity between the two enzymes is observed only when the helicase is located within the influence range of the DNAP that extends to two downstream nucleotides on the template. The helicase follows the fork junction; therefore, cooperative base pair melting is observed only when the helicase is located one nucleotide ahead of the DNAP. This provides the precise positions of the two enzymes at the replication fork junction.
Based on the new structural insights on the positions of the helicase and DNAP combined with the kinetic studies that identified the rate-limiting steps, I propose a detailed model of helicase-DNAP at the leading strand replication fork. This model explains how helicase-DNAP unwind-synthesize dsDNA independent of the GC content and at rates that resemble translocation on ssDNA.