Identification of a New Motif Required for the 3′–5′ Exonuclease Activity of Escherichia coli DNA Polymerase I (Klenow Fragment)

Abstract

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...

Curing of mixtures of epoxy resins and 4‐methyl‐1,3‐dioxolan‐2‐one with several initiators

“…This implies that the backtracking long pause is usually associated with the proofreading, which is consistent with the experimental data 10, 29, 34. This proofreading associated with the backtracking pause for RNAP corresponds to that associated with the 3′–5′ exonuclease activity for high‐fidelity DNA polymerase (DNAP),53, 54 and the fidelity resulting from the nucleotide insertion checkpoint55 is not considered here.…”

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

“…In contrast the binding of ssDNA to Klenow was relatively weak, with an apparent K d of about 40 nM. This is very close to previously reported estimates of K d for ssDNA binding to Klenow in similar buffer conditions (36). We conclude that a significant component of the binding free energy of P/T junction DNA to Klenow is contributed by interactions involving the double-stranded portion of the construct.…”

“…The protein residues interacting with the DNA in these sites are clearly different for the pol and exo mode-bound complexes (36,40). In this study of polymerase-DNA complexes in solution, we have characterized the local conformations of bases interacting with the first three polymerase binding sites listed above.…”

Structural Basis for the dsRNA Specificity of the Lassa Virus NP Exonuclease

“…These exonucleases contain a basic loop motif adjacent to the active site that may facilitate melting of duplex DNA in order to provide a suitable single-stranded 3′ end for the active site [6] , [9] , [19] . Certainly, mutation of this basic loop in KF and Trex2 reduces exonuclease activity [9] , [21] , suggesting a role for the motif in binding nucleic acid, particularly the non-substrate strand as it extends from the active site [9] , [21] . We note that in Lassa NP, the site that is positionally equivalent to the basic loop of the other exonucleases is neither basic nor loop-shaped, but is instead much shorter and mostly hydrophobic ( Figure 7 ).…”