Loss of DNA Polymerase β Stacking Interactions with Templating Purines, but Not Pyrimidines, Alters Catalytic Efficiency and Fidelity

Abstract

Structures of DNA polymerases bound with DNA reveal that the 5-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, ␣-helix N of DNA polymerase ␤ is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions. DNA polymerase (pol)1 ␤ is an attractive model to study polymerase strategies employed to assure efficient and faithful DNA synthesis. Its small size (39-kDa) and lack of accessory proteins facilitates its biochemical, kinetic and structural characterization. DNA polymerase ␤ is also the only eukaryotic polymerase for which there is a high-resolution crystal structure. It is structurally and kinetically suited to function on short DNA gaps during DNA repair or replication (1). Although pol ␤ (pol X family) appears to have evolved separately from other families of polymerases of known structure (2), it shares many general structural and mechanistic features with these polymerases. The polymerase domain is composed of three functionally distinguishable subdomains. The polymerase catalytic subdomain binds two divalent metal cations that assist the nucleotidyl transferase reaction. Two additional subdomains that have primary roles in duplex DNA binding and nucleoside 5Ј-triphosphate (dNTP) selection surround the catalytic subdomain. These subdomains will be referred to as C (catalytic), D (duplex DNA binding), and N (dNTP selection) subdomains to discern their intrinsic function. These would correspond to the palm, thumb, and fingers subdomains, respectively, according to the nomenclature that utilizes the architectural analogy to a right-hand (3). 2DNA polymerase ␤ also utilizes a kinetic mechanism similar to other polymerases. Steady-state and pre-steady-state kinetics analyses indicate that DNA polymerases follow an ordered binding of substrates, with DNA ...

Influence of DNA Structure on DNA Polymerase β Active Site Function

“…Kinetic Assays-Steady-state kinetic parameters for single-nucleotide gap-filling reactions were determined by initial velocity measurements as described previously (13). Unless noted otherwise, enzyme activities were determined using a standard reaction mixture (50 l) containing 50 mM Tris-HCl, pH 7.4, 100 mM KCl, 5 mM MgCl 2 , and 200 nM single-nucleotide gapped DNA.…”

“…In such a situation, K m is equivalent to K d for the incoming nucleotide (25). The K d for the correct incoming nucleotide on a matched terminus is ϳ10 M (11,13). Thus, the increase in K m for these mismatches reflects the diminished k pol and not a lower binding affinity for the incoming nucleotide.…”

“…Lys 280 and Asp 276 of ␣-helix N contribute van der Waals interactions with the templating and incoming nucleotide bases, respectively, whereas Asn 279 and Arg 283 contribute DNA minor groove interactions. Structure-based sitedirected mutagenesis of these residues has identified interac-tions that contribute to efficient DNA synthesis (7)(8)(9)(10)(11)(12)(13). The specific contribution provided by these interactions appears to be dependent on the identity of the base pair that is formed.…”

“…The specific contribution provided by these interactions appears to be dependent on the identity of the base pair that is formed. For example, decreasing the stacking interactions of residue 280 with the templating base by site-directed mutagenesis resulted in a more dramatic loss in binding affinity for incoming complementary pyrimidines than purines, indicating that the energetic contributions of specific side chain interactions are strongly dependent on the specific insertion (13).…”

Incorrect nucleotide insertion at the active site of a G:A mismatch catalyzed by DNA polymerase β

“…Earlier studies had found that the rate of catalytic reaction for the correct base pair is a crucial factor in achieving high fidelity in DNA synthesis (7). The incorrect nucleotide insertion reaction catalyzed by DNA polymerases exhibiting modest to high fidelity is much slower than for the correct insertion (8,9).…”