Crystal structures of a ddATP-, ddTTP-, ddCTP-, and ddGTP-trapped ternary complex of Klentaq1: Insights into nucleotide incorporation and selectivity

The mechanism by which DNA polymerase I enzymes function has been the subje ct of extensive biochemical and structural studies. We previously determine d the structure of a ternary complex of the large fragment of DNA polymeras e I from Thermus aquaticus (Klentaq1) bound to a primer/template DNA and a dideoxycytidine 5'-triphosphate (ddCTP), In this report, we present the det ails of the 2.3-Angstrom resolution crystal structures of three additional ternary complexes of Klentaq1 bound to a primer/template DNA and a dideoxyg uanosine 5'-triphosphate (ddGTP), a dideoxythymidine 5'-triphosphate (ddTTP ), or a dideoxy adenosine 5'-triphosphate (ddATP), Comparison of the active site of the four ternary complexes reveals that the protein residues aroun d the nascent base pair (that formed between the incoming dideoxynucleoside triphosphate [ddNTP] and the template base) form a snug binding pocket int o which only a correct Watson-Crick base pair can fit. Except in the ternar y complex bound to dideoxyguanosine 5'-triphosphate, there are no sequence specific contacts between the protein side chains and the nascent base pair , suggesting that steric constraints imposed by the protein onto the nascen t base pair is the major contributor to nucleotide selectivity at the polym erase active site, The protein around the polymerase active site also shows plasticity, which may be responsible for the substrate diversity of the en zyme. Two conserved side chains, Q754 and R573, form hydrogen bends with th e N3 atom in the purine base and O2 atom in the pyrimidine base at the mino r groove side of the base pair formed by the incorporated ddNMP and the cor responding template base in all the four ternary complexes. These hydrogen- bonding interactions may provide a means of detecting misincorporation at t his position.