The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

DNA polymerase is the central component of replicases that are responsible for faithfully copying DNA. Despite the fact that all four dNTPs are potential substrates, replicative DNA polymerases are able to limit the incorporation of mismatched bases to about one in a million [for reviews see (1–8)]. Although this error frequency is acceptable for phage replication, it is still far too high to maintain genetic integrity during cell proliferation in more complex organisms. To increase fidelity, cells have a number of mechanisms for correcting errors, including the ability of the replicative DNA polymerase itself, or an associated subunit, to excise misincorporated bases. In addition, there are specialized DNA polymerases that confront DNA damage inflicted by radiation, oxidation, alkylating agents, etc. which block the progress of replicative polymerases (6, 9–20). These DNA polymerases, as exemplified by members of the Y family, are less selective toward incoming dNTPs and have lower catalytic efficiencies for correct insertion compared with replicative polymerases (21, 22). On the other hand, misincorporation results in reduced primer extension rates, providing an opportunity for the exonuclease activity of the replicative polymerases or their associated subunits, to remove mismatched bases at the 3’ primer-terminus of the primer before they become part of the DNA duplex (23, 24). Because the rate of extension beyond a mismatch is also slower than continued DNA synthesis from a correctly base paired primer-template terminus, the opportunity for error correction by the exo activity of the polymerase or replicase is further increased (24). In the continuing quest to understand the molecular basis for base discrimination exhibited by replicative DNA polymerases, we have chosen to study the DNA polymerase from bacteriophage RB69. RB69 pol, like its closely related homolog T4 DNA polymerase, belongs to the B family and has sequence similarities with many eukaryotic DNA pols such as human pol α (25). The crystal structures of the Apo form of the RB69 pol and several RB69 pol-DNA complexes have been solved (12, 26–29). The palm, fingers and thumb domains, which are clearly recognizable in RB69 pol, are common features shared by many DNA polymerases. The catalytic center for the phosphoryl transfer reaction lies in the palm, the most highly conserved domain among all the polymerases (30). Amino acid residues that comprise the nascent base-pair binding pocket of RB69 pol in the “closed” conformation have been identified and are shown in Figure 1 (29). The interactions and putative function of the side chains of these residues with the incoming dNTPs are as following: (i) Y416 functions as a “sugar gate” to distinguish between the ribose and the deoxyribose moieties associated with the incoming nucleoside triphosphate (31); (ii) K560 interacts with the non-bridging α and γ oxygens in the triphosphate moiety of the incoming dNTP (32); (iii) the hydroxyl group of residue Y567 forms a hydrogen bond with the minor groove edge of the DNA duplex at the primer-template terminus (29). Replacement of the aromatic side chain of Y567 with a methyl group (Y567A) reduces base discrimination against mismatched bases without affecting the rate of incorporation for complementary dNMPs (33, 34). As part of this nascent base-pair binding pocket, L415, Y416, and Y567 are involved in hydrophobic interactions with the minor groove of the nascent base pair. The rear wall of this binding pocket, which includes residues L561, N564 and S565 as shown in Fig. 3 of (12), is presumed to reinforce the coplanar arrangement of the nascent base pair (12). According to the ternary structure of RB69 pol (29), the side chain of L561 is an obvious protrusion into the nucleotide binding pocket that may clash with a bulge on the major groove side of a mispair. Even though this steric clash is estimated to cause only about 0.1 to 0.2A of overlap, it may be sufficient to account for the selectivity against purine-purine mismatches, since these mispairs need more space than is available in the binding pocket. The amino acid residues cited above define a very tight binding pocket that has evolved to accommodate Watson-Crick base pairs but leaves little space for water molecules or for bulges caused by incorrect geometry of non-complementary base pairs. Figure 1 Space-filling model of residues in the fingers and palm domains that are part of the nascent base-pair binding pocket (29). Included are space-filling models of dTTP (gold) pairing with a templating A (grey). The structure of an A-form A-C mismatch (magenta ... Figure 3 Structural basis for discrimination against purine-pyrimidine mismatches. Modeling of the dA-C (cyan) and dC-A (magenta) mismatches into the ternary RB69 pol structure (29) was carried out using four atoms defining two glycosidic bonds, because all purine-pyrimidine ... We have previously reported that the RB69 pol Y567A variant exhibits reduced base selectivity and enhanced mutagenicity compared to the wild-type RB69 pol (33, 34). Here we present in vivo and in vitro data on the consequences of replacing L561 with Ala. We also discuss the frequency and types of errors that the RB69 pol L561A variant generates in vivo. In addition, the kinetic parameters for the wild type enzyme and L561A variant have been determined in vitro under single-turnover conditions with matched and all combinations of mismatched incoming dNTPs. We compared our results with those found for the Klenow fragment (KF), where similar types of studies have been carried out (35, 36). We have also placed various non-complementary base-pairs into the RB69 pol ternary complex in silico to see if the kinetic data from the wild type and the L561A variant polymerase, with the mispaired bases, could be rationalized by modeling.