Mechanisms of Fidelity and Mutagenesis

Project: Research project

Project Details

Description

DESCRIPTION: (PROVIDED BY APPLICANT) The long-term goal of this project is to
understand the chemical interactions that govern replication of DNA. The
short-term object of this research proposal is to determine the role that
hydrogen bonding, between the polymerase and the minor groove of the DNA, plays
in the fidelity of DNA replication. The critical interactions that control the
fidelity of DNA replication occur in the transition states of several of the
individual steps that comprise DNA synthesis. The transition states will be
probed by structure activity relationships. The structure of the reactants will
be altered by atomic substitution of the DNA and amino acid substitution of the
polymerases, and the progress of the reaction will be measured with
pre-steady-state kinetics. Two polymerases will be studied, DNA polymerase I of
E. coli and mammalian DNA polymerase beta. The biochemical mechanisms of DNA
synthesis by these proteins have been well studied, thus providing a firm
foundation for structure-function analyses. Moreover, as the enzymes display
structural differences, they may utilize different mechanisms for fidelity
control. The following specific hypotheses will be tested. (1) Hydrogen bonds
between polymerase and the minor groove of DNA are crucial to catalysis and
fidelity of DNA replication. In particular, Arg283 of polymerase beta makes a
crucial hydrogen bond to the N3-position of a purine of the template base. (2)
Arg668 of E. coli DNA polymerase I acts as a sensor for the correct geometry at
the terminal base pair through interactions with N3 of guanine and ring oxygen
of the incoming dNTP. (3) The interactions between polymerases and the minor
groove of DNA are dependent on the sequence of the DNA. (4) The interactions
between polymerases and the O2-positions of dTTP and dCTP are important due to
steric interactions between the nucleotide and the protein. (5) The low
fidelity bypass polymerases use minor groove interactions to replicate DNA. The
results of these experiments will provide very specific mechanistic information
of how polymerases replicate DNA with high fidelity. This knowledge will add to
our fundamental knowledge of DNA replication.
StatusFinished
Effective start/end date7/16/9712/31/07

Funding

  • National Cancer Institute: $243,556.00
  • National Cancer Institute: $261,013.00
  • National Cancer Institute: $261,013.00
  • National Cancer Institute
  • National Cancer Institute
  • National Cancer Institute: $75,745.00
  • National Cancer Institute
  • National Cancer Institute: $261,013.00

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