Mechanisms of Fidelity and Mutagenesis

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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.