DNA mismatch synthesis complexes provide insights into base selectivity of a B family DNA polymerase.

摘要: Current hypotheses that attempt to rationalize the high degree of base selectivity exhibited by replicative DNA polymerases (pols) concur ternary complexes formed with incorrect dNTPs are destabilized. Knowing what accounts for this destabilization is likely be key understanding discrimination. To address issue, we have determined crystal structures all 12 mismatches using an engineered RB69 pol quadruple mutant (qm, L415A/L561A/S565G/Y567A) enabled us capture nascent mispaired dNTPs. These show in base-pair binding pocket (NBP) qm differ markedly from embedded binary pol-DNA complexes. Surprisingly, only 3 clash NBP when they modeled into wild-type (wt) pol. The remaining can fit a wt complex but deviate normal Watson-Crick base-pairs. Repositioning templating nucleotide residue and enlarged play important roles accommodating incoming From these structures, propose additional reasons as why incorporated so inefficiently pol: (i) steric clashes side chains after Fingers closing; (ii) weak interactions or large gaps between dNTP base; (iii) burying protonated hydrophobic environment NBP. All possibilities would expected destabilize closed incorporation rare event.