Cocrystal structure of an editing complex of klenow fragment with dna (3'-5' exonuclease dna polymerase protein-dna interaction x-ray crystallography/metal ion catalysis)
High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3′ – 5′ exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3′ terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 Å away for the newly formed 3′ terminus. Since a 3′ terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.
