Using a fluorescent cytosine analogue tC(o) to probe the effect of the Y567 to Ala substitution on the preinsertion steps of dNMP incorporation by RB69 DNA polymerase.

Replication of a genome from any organism must occur with minimal mistakes if the progeny are to remain viable. Replicative DNA polymerases perform this task, making less than one mistake per 106 base-pairs synthesized.(1–4) Fidelity is increased by an additional 100-fold if the polymerase has exonuclease capability.(5–7) The number of errors made during replication depends on how well a DNA polymerase (pol) can recognize a dNTP as either correct or incorrect for pairing opposite the templating base. Sometimes a DNA pol can circumvent the geometric constraints imposed by residues in the Nascent Base-pair binding Pocket (NBP) so that incorrect dNTPs can be incorporated albeit with low efficiency. Based on crystal structures and studies using fluorescent probes, selection of the correct dNTP most likely occurs prior to phosphoryl transfer.(8–16) Subsequent to dNTP binding (Scheme 1), the polymerase Fingers domain closes, aligning the reactive centers of the substrates, leading to rapid nucleotidyl transfer.(13, 17–19) The incorrect dNTP either fails to induce Fingers closing, or if the Fingers do close the result is an unstable, but potentially productive, ternary complex.(13, 20–21) Scheme I Pathway of dNTP binding and incorporation. EDn is the open complex, and FDn is the closed complex. DNA pols from different families, and even within families, differ greatly in their ability to resist insertion of incorrect dNTPs. The B family DNA pol from bacteriophage RB69 (RB69pol) maintains high fidelity even when it encounters damaged templates or templating base analogues.(22–28) Interestingly, upon substitution of Y567 with Ala in the NBP, the mutant RB69pol inserts incorrect dNTPs with efficiencies 102 to 103 fold greater than wild type (wt) pol.(29) Crystal structures of ternary complexes of the RB69pol Y567A mutant suggest that the reduction in base selectivity exhibited by this mutant was likely due to an increase in NBP flexibility, leading to stable conformations of the templating base even when paired opposite incorrect dNTPs, a situation that would otherwise be blocked in the wt enzyme.(15, 29) However, a fuller understanding of how increased flexibility of residues in the NBP affects base selectivity requires analysis of kinetic steps that occur prior to nucleotidyl transfer. For this purpose we employed the highly fluorescent 1,3-diaza-2-oxophenoxazine (tC°) to probe the pre-insertion steps before chemistry. As shown in Fig 1, tC° is a tricyclic cytosine analogue. When presents as the templating base, it can be used to follow isomerization of DNA pols from an open to a closed conformation in response to binding of an incoming dNTP.(12, 14) Furthermore, unlike 2-aminopurine (2AP), a well-characterized fluorescent adenine analogue, where fluorescence quenching is totally dependent on base stacking, quenching of tC° fluorescence depends on the formation of hydrogen bonds (HB) with the templating base.(30–34) Sandin et al reported that tC° base pairs exclusively with guanine (G) and caused minimal perturbations in the structure of duplex DNA.(35–36) Kuchta's lab has recently discovered that tC° triphosphate (dtC°TP) is preferentially inserted opposite templating G and A residues by both KF and human DNA pol alpha.(37) They speculated that the incorporation of dAMP opposite tC° and vice-versa was due to the propensity of tC° to tautomerize into its imino form (Fig. 1).(37) Figure 1 Pairing of guanine (G) and adenine (A) opposite tC°. The analogue pairs with adenine upon tautomerization to the imino form. We found that tC° fluorescence was quenched only when paired opposite dGTP in a wt RB69pol ternary complex, reflecting formation of a stable nascent base-pair.(15) Surprisingly, we found that the net equilibrium dissociation constant (Kd,net) for dGTP binding opposite tC° with the Y567A mutant, in the absence of chemistry, was reduced by more than 200 fold relative to the corresponding wt ternary complex. In addition, dATP was able to form a stable nascent base-pair with tC° only with the Y567A mutant. To gain further insight into why this mutant has increased affinity for dGTP and was permissive for dATP binding, we determined the crystal structures of the dGTP/tC° and dATP/tC°-containing ternary complexes of the RB69pol Y567A mutant at 1.9 A resolution. We found that incoming dATP formed two HBs with an imino-tautomerized form of tC°. However, stabilization of the dATP/tC° base-pair required: (i) movement of the tC° backbone sugar into the DNA minor groove; (ii) lateral shifting of residues A567 and G568 toward residue Y416; (iii) tilting of the tricyclic ring of tC° base to avoid a steric clash with L561. Together, these data suggest that flexibility of the NBP provided by replacement of Y567 with Ala, led to a more favorable isomerization step (higher k2 value compared to the k2 exhibited by wt pol, as shown in Scheme 1) resulting in an increase in substrate binding affinity.