DNA polymerases play a crucial role in the cell cycle due to theirinvolvement in genome replication and repair. Errors in replication canproduce mutations that can lead to disease or even death. Thus, understandingthe molecular mechanism for correct base pairing can lead to insights into themutagenesis process. Recent single--molecule F�rster resonance energytransfer (smFRET) studies on DNA polymerase I (Klenow Fragment) (KF) haverevealed a previously unknown step in DNA polymerization.  Following eachnucleotide incorporation, KF changed conformation in a process consistentwith a 2--base downstream movement along the template before returning tothe proper location (1--base downstream) for the next base incorporation. Theseresults have led to the hypothesis of a post--insertion fidelity--checkingsite that ensures proper base paring as part of the proofreading mechanism.To help test this hypothesis, we have used energy analysis decomposition (EDA)to identify residues involved in this checking site. To this end, MD simulations were carried out for T--A (correctly paired) and threemispairs (C--A, G--A and A--A) DNA trimers in the pre--insertionand putative checking sites in KF. The structures generated from these runswere subjected to the EDA. The results show that several residues have alterednon--bonded interactions for the correctly paired DNA trimer compared to themispairs.  All these residues are located roughly within 12 � of theputative checking site. Furthermore, a sequence alignment of A family DNApolymerases shows that 7 of the residues obtained from the EDA are totallyconserved across the family and across species. These residues providepotential targets for mutagenesis studies.

Computational determination of residues for nucleotide misincorporation discrimination in <i>E. coli</i> DNA polymerase I

239th National Meeting (2010)

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Computational determination of residues for nucleotide misincorporation discrimination in E. coli DNA polymerase I