VISUALIZING DNA-REPLICATION IN A CATALYTICALLY ACTIVE BACILLUS DNA-POLYMERASE CRYSTAL

DNA polymerases copy DNA templates with remarkably high fidelity, chec king for correct base-pair formation both at nucleotide insertion and at subsequent DNA extension steps(1-3). Despite extensive biochemical, genetic and structural studies(2,4), the mechanism by which nucleotid es are correctly incorporated is not known, Here we present high-resol ution crystal structures of a thermostable bacterial (Bacillus stearot hermophiIus) DNA polymerase large fragment(5) with DNA primer template s bound productively at the polymerase active site. The active site re tains catalytic activity, allowing direct observation of the products of several rounds of nucleotide incorporation, The polymerase also ret ains its ability to discriminate between correct and incorrectly paire d nucleotides in the crystal, Comparison of the structures of successi vely translocated complexes allows the structural features for the seq uence-independent molecular recognition of correctly formed base pairs to be deduced unambiguously. These include extensive interactions wit h the first four to five base pairs in the minor groove, location of t he terminal base pair in a pocket of excellent steric complementarity favouring correct base-pair formation, and a conformational switch fro m B-form to underwound A-form DNA at the polymerase active site.