KINETIC ANALYSIS OF THE HELICASE-NTPASE

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DESCRIPTION: DNA helicases are ubiquitous proteins that are required in vivo for unwinding duplex DNA into single-stranded DNAs, a process energetically coupled to NTP hydrolysis. DNA helicases are important in all processes of DNA metabolism and a defect in DNA helicase has been found to be responsible for human diseases such as xeroderma pigmentosa, cockayne's syndrome, and bloom syndrome. The long term goal of the research is to understand the mechanism of this energy transducing enzyme at the kinetic, thermodynamic, and structural level. The investigators have chosen bacteriophage T7 DNA helicase to study as a model system. T7 DNA helicase is the primary helicase of T7 bacteriophage involved in DNA replication. It is a ring-shaped hexamer of identical subunits that has a central hole through which it binds ssDNA. Each hexamer binds only 3 NTPs, and the equilibrium DNA binding studies by the investigator have shown that the helicase interacts with the DNA more tightly in the "NTP-state" vs. the "NDP-state". The ssDNA binds asymmetrically to the hexamer interacting with only one or two subunits at any given time. The investigators propose that NTP is hydrolyzed by the helicase hexamer in a coordinated manner that leads to "DNA bind-release" required for translocation of the helicase on the DNA. In this model, each monomer or dimer interacts with the DNA in a sequential manner to catalyze translocation required for DNA unwinding. The NTPase reaction provides the "switch" for the DNA bind-release process. Experiments are proposed to test and distinguish between various mechanisms by measuring the single-turnover kinetics of NTP and DNA binding and the presteady state kinetics of the NTPase reaction. It is known that gp4 helicase requires two noncomplementary ssDNA tails at one end of the duplex DNA (fork DNA) to initiate DNA unwinding. The first step to understanding the unwinding mechanism is to determine the interaction of the helicase with the fork DNA. The investigators propose experiments to study the role of the 3'-tail in DNA unwinding, and determine the rate of DNA unwinding. Studies are proposed with the following specific aims: i) to measure the kinetics of nucleotide and DNA binding by stopped-flow methods, ii) to measure the presteady state kinetics of dTTP hydrolysis by rapid chemical quench-flow to understand the coordination in dTTP binding, its hydrolysis, and product dissociation by the subunits of the hexamer, and iii) to investigate the role of the 3'-ssDNA tail, and measure the intrinsic rate of DNA unwinding.