KINETIC ANALYSIS OF THE HELICASE-NTPASE

Project Details

Description

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.
StatusFinished
Effective start/end date1/1/977/31/17

Funding

  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $274,243.00
  • National Institute of General Medical Sciences: $266,900.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $302,321.00
  • National Institute of General Medical Sciences: $289,230.00
  • National Institute of General Medical Sciences: $302,321.00
  • National Institute of General Medical Sciences: $341,321.00
  • National Institute of General Medical Sciences: $317,850.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $282,433.00
  • National Institute of General Medical Sciences: $266,900.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $266,900.00
  • National Institute of General Medical Sciences: $170,880.00
  • National Institute of General Medical Sciences: $314,672.00
  • National Institute of General Medical Sciences: $266,900.00
  • National Institute of General Medical Sciences: $245,077.00
  • National Institute of General Medical Sciences: $311,525.00
  • National Institute of General Medical Sciences: $274,243.00
  • National Institute of General Medical Sciences: $67,726.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Genetics
  • Molecular Biology
  • Structural Biology
  • Filtration and Separation

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