The reopening rate of the fingers domain is a determinant of base selectivity for RB69 DNA polymerase.

DNA polymerase is the central component of a multiprotein complex (replicase) that is responsible for faithfully copying the genomes of all living organisms. Although all four deoxynucleoside triphosphates are potential substrates, replicative DNA polymerases insert “wrong” bases only once in every 1 million incorporation events. The main reason for the low error rate is the high level of base discrimination exhibited by these enzymes (for reviews, see refs 1–4). If the wrong base is inserted, it is quickly removed via a potent exonuclease activity either inherent in the polymerase polypeptide or in a subunit associated with the replication complex which increases the overall fidelity by at least 100-fold (5–11). High on the list among the outstanding unsolved problems in DNA replication is the mechanism used by DNA polymerases to discriminate against three of the four dNTPs that do not form Watson–Crick (W–C) hydrogen bonds with the templating base. As has been noted many times, the difference in free energy, between base pairs that can form W–C hydrogen bonds and those that cannot, is insufficient to account for the degree of base selectivity that has been observed with replicative DNA polymerases (3, 12). In this paper, we report results of kinetic studies with RB69 DNA polymerase (RB69 pol)1 that provide some insight into this process. In the preceding paper (13), we investigated the effect that occupancy of either the “A” or “B” metal ion site would have on (i) the affinity of an incoming dNTP, (ii) the rate of conformational change, and (iii) the rate of primer extension. This was possible because we took advantage of the “exchange-inert” properties of the rhodium(III) cation (Rh3+) (14). Since the Rh3+ cation forms stable complexes with deoxynucleoside triphosphates (14), we were able to saturate the B metal ion site and the nucleotide binding pocket by adding a sufficient Rh·dTTP concentration to the RB69 pol–dP/T binary complex while leaving the A metal site unoccupied. We showed that the RB69 pol–dP/T–Rh·dTTP collision complex could proceed to a closed form even when the A metal ion site was empty. In the absence of Mg2+ or Mn2+ in the A site, chemistry does not take place. However, we wanted to know whether Mg2+ can enter the closed form of the ternary structure to allow the nucleotidyl transfer reaction to occur. If there were no barriers to free diffusion of Mg2+ into the A site in the closed ternary complex, then the rate of nucleotide incorporation would be expected to equal the rate that was observed when Mg2+ was included in the reaction mixture before addition of Rh·dTTP. On the other hand, if the closed structure has to relax, at least partially to a more open form, before Mg2+ can access the A site, then it should be possible to determine the rate of relaxation, providing that this process is slower than the rate of nucleotidyl transfer. The rate of relaxation or reopening corresponds to k−3 in the minimal kinetic scheme (Scheme 1) for the nucleotidyl transfer reaction catalyzed by RB69 pol as shown below: Scheme 1 The closed ternary structure is designated as FDN because it is in a fluorescent state different from that of the EDN collision complex that forms upon introduction of a dNTP(N) to an RB69 pol–dP/T(ED) complex where a 2-aminopurine (dAP) residue is the templating base. Previous studies have shown that when a saturating dTTP concentration is introduced into a solution of this type of binary complex, the fluorescence of dAP is rapidly quenched at a rate, k+3, that exceeds the rate of dTMP incorporation (15, 16). When all of the ternary complexes are in the FDN state, chemistry occurs at the maximal rate (k+4) and is essentially irreversible. In this report, we show that when Mg2+ is excluded from the reaction until the ternary complex is formed, the rate of dTMP incorporation is much slower (4 s−1) than when Mg2+ is present at the time when the components are first mixed (85 s−1). We propose that the 4 s−1 rate represents the reopening rate of the ternary complex that can be observed by rapid chemical quench and limits the rate of dTMP incorporation under these circumstances. The other means of estimating the reopening rate are indirect and rely on the rate of dAP fluorescence enhancement when dTTP is released from an RB69 pol–dP/T–dNTP ternary complex. As discussed later, an experiment designed to determine the reopening rate of the ternary complex by trapping dTTP as it dissociates from the ternary complex revealed two rates of fluorescence enhancement with equal amplitudes, 50 and 4 s−1. These determinations were made with the correct incoming dNTP. As we show later when an incorrect dNTP is part of a ternary complex, the reverse isomerization (reopening) rate is very rapid. Given these results, the question is whether they provide any additional insight into how RB69 pol is able to exert such a high level of base discrimination. There have been proposals published that ascribe base selectivity to differences in the relative energy barriers of the transition state for the incorporation of correct and incorrect bases (11, 12, 17–19). Other schemes to account for base discrimination invoke a rate-limiting step (conformational change) that acts as a checkpoint before chemistry (2, 3, 20). Recently, a kinetic explanation was advanced which stated that base discrimination is dependent on the relative ratio of the nucleotidyl transfer rate (k+4) to the rate of reopening (k−3) of ternary complexes that contain either a correct or an incorrect dNTP (21). We decided to test this latter proposal by attempting to estimate the reversal rate (k−3) of FDN, the closed, isomerized RB69 pol ternary complex, a state that exists just prior to the nucleotidyl transfer step, as shown in Scheme 1. As mentioned above, we have used pulse–chase, chemical-quench and stopped-flow fluorescence procedures for this purpose. We found that the value of k−3 was much smaller than k+4, the rate of chemistry, for insertion of correct incoming dNTPs but is very rapid for incorrect dNTPs. As a consequence, only a small percentage of ternary complexes with an incorrect dNTP populate the FDN state. A low flux of incoming nucleotides through the FDN state would account for the dramatically reduced efficiency for incorporation of incorrect bases into the growing primer strand and is consistent with the proposal of Tsai and Johnson (21) for base selectivity exhibited by T7 pol. The implications of this hypothesis for the fidelity of RB69 pol and other B family replicative DNA polymerase are discussed.