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| - Fidelity of Mispair Formation and Mispair Extension Is Dependent on theInteraction between the Minor Groove of the Primer Terminus and Arg668 of DNAPolymerase I of Escherichia coli
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| - The hydrogen bonding interactions between the Klenow fragment of Escherichia coli DNApolymerase I with the proofreading exonuclease inactivated (KF-) and the minor groove of DNA wereexamined with modified oligodeoxynucleotides in which 3-deazaguanine (3DG) replaced guanine. Thissubstitution would prevent a hydrogen bond from forming between the polymerase and that one site onthe DNA. If the hydrogen bonding interaction were important, then we should observe a decrease in therate of reaction. The steady-state and pre-steady-state kinetics of DNA replication were measured with 10different oligodeoxynucleotide duplexes in which 3DG was placed at different positions. The largestdecrease in the rate of replication was observed when 3DG replaced guanine at the 3‘-terminus of theprimer. The effect of this substitution on mispair extension and formation was then probed. The G to3DG substitution at the primer terminus decreased the kpol for the extension past G/C, G/A, and G/G basepairs but not the G/T base pair. The G to 3DG substitution at the primer terminus also decreased theformation of correct base pairs as well as incorrect base pairs. However, in all but two mispairs, the effecton correct base pairs was much greater than that of mispairs. These results indicate that the hydrogenbond between Arg668 and the minor groove of the primer terminus is important in the fidelity of bothformation and extension of mispairs. These experiments support a mechanism in which Arg668 forms ahydrogen bonding fork between the minor groove of the primer terminus and the ring oxygen of thedeoxyribose moiety of the incoming dNTP to align the 3‘-hydroxyl group with the α-phosphate of thedNTP. This is one mechanism by which the polymerase can use the geometry of the base pairs to modulatethe rate of formation and extension of mispairs.
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