Insights into DNA polymerase 's mechanism for accurate DNA replication

Our study examines the mechanisms by which DNA polymerase (pol) faithfully replicates DNA. To better understand this process, we have performed all-atom molecular dynamics simulations of several DNA pol systems to identify conformational changes occurring prior to chemistry and investigate mechanisms by which mutations in the fingers domain (R696W and A699Q) lower fidelity. Our results indicate that, without the incoming nucleotide, a distinct open conformation occurs defined by a rotation in the fingers. The closed form, adopted when the correct nucleotide is bound, appears best organized for chemistry when three magnesium ions coordinate protein and DNA residues in the active site. Removing an unusual third metal ion from the polymerase active site causes shifting in the fingers and thumb as well as stimulating specific exonuclease -hairpinDNA interactions that fray the primer terminus base pair. These changes suggest that dissociation of the third divalent ion (metal ion C') signals a transfer of the DNA primer from the polymerase to the exonuclease active site and implies a role for the -hairpin in DNA switching. Analysis of -hairpin movement in several systems reveals a dependence on active-site changes and suggests how Lys444 and Tyr446 present in the -hairpin can affect proofreading. Analysis of A699Q and R696W pol mutant systems reveal marked differences in the open-to-closed transition as well as -hairpin repositioning that explain reduced nucleotide selectivity and higher error rates.