The Klenow fragment of Escherichia coli DNA polymerase I houses catalytic centers for both polymerase and 3 -5 exonuclease activities that are separated by about 35 Å . Upon the incorporation of a mismatched nucleotide, the primer terminus is transferred from the polymerase site to an exonuclease site designed for excision of the mismatched nucleotides. The structural comparison of the binary complexes of DNA polymerases in the polymerase and the exonuclease modes, together with a molecular modeling of the template strand overhang in Klenow fragment, indicated its binding in the region spanning residues 821-824. Since these residues are conserved in the "A" family DNA polymerases, we have designated this region as the RRRY motif. The alanine substitution of individual amino acid residues of this motif did not change the polymerase activity; however, the 3 -5 exonuclease activity was reduced 2-29-fold, depending upon the site of mutation. The R821A and R822A/ Y824A mutant enzymes showed maximum cleavage defect with single-stranded DNA, mainly due to a large decrease in the ssDNA binding affinity of these enzymes. Mismatch removal by these enzymes was only moderately affected. However, data from the exonuclease-polymerase balance assays with mismatched template-primer suggest that the mutant enzymes are defective in switching mismatched primer from the polymerase to the exonuclease site. Thus, the RRRY motif provides a binding track for substrate ssDNA and for nonsubstrate single-stranded template overhang, in a polarity-dependent manner. This binding then facilitates cleavage of the substrate at the exonuclease site.The faithful synthesis of DNA by E. coli DNA polymerase I (pol I) 2 is accomplished by a combination of two processes: (a) correctly incorporating matched nucleotides and (b) removing incorrectly incorporated mismatched nucleotides with its 3Ј-5Ј exonuclease activity (1). The incorporation of a mismatched nucleotide on a growing primer strand stalls further DNA synthesis and decreases the affinity of the polymerase for the template-primer. This results in shuttling of the primer to the exonuclease active site (exo site). Thus, error correction by DNA polymerases that contain both the polymerase and the exonuclease activities involves at least the following four steps: (a) sensing of the misincorporated nucleotide at the polymerase site (pol site), (b) transfer of the mismatched primer from the pol to the exo site, (c) excision of the incorrect nucleotide, and (d) transfer of the corrected primer terminus to the pol site for further nucleotide incorporation. The mechanistic details of these steps are not fully understood. However, the availability of DNA polymerase structures, with DNA bound in either the polymerase or the exonuclease mode, provides some clues to the possible structural alterations of the polymerase protein during DNA synthesis.The active site of DNA polymerases catalyzing the polymerase reaction has a configuration that allows non-sequence-specific contacts with both DNA and the incom...

The Klenow Fragment (KF) of E. coli DNA polymerase I has two activities on a single polypeptide chain‐the polymerase and the 3′‐5′exonuclease. These activities collectively function to enhance an error‐free synthesis of DNA. The active sites for the two activities however are separated by a distance of ~30Å. Thus, a movement of primer strand containing terminal mismatch would require 30Å movement. This may further involve repositioning single stranded template overhang to facilitate the movement. In order to understand the structural elements that are involved in coordination between polymerase and the exonuclease activity we examined crystal structures of KF and related enzymes with the primer bound in either exonuclease or polymerase mode. Superposition of the two structures revealed that template overhang requires to shift its position by ~ 20Å and that it is in the close vicinity of positively charged residues. These residues most likely bind to template overhang and permit primer strand motion to the exonuclease site. Because of the conservation of this cluster in a number of pol I related enzymes we have named it as R/KRRY motif. To assess this postulate, we generated mutants of the residues R822A, Y824A and a double mutant R822AY824A. Preliminary examination of the properties of these mutant enzymes has shown that the polymerase activity is relatively unchanged but their exonuclease activity on ssDNA and DNA with 1 and 3 mismatches is significantly reduced (4–14 fold reduction). Biochemical characterization of the mutant enzymes is in progress to determine the defective step. This study would establish the role of the R/KRRY motif residues in stabilizing the template strand when the primer binds in the exonuclease mode. Support: NIGMS36307