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

by
Pinky Kukreti
Biochemistry and Molecular Biology

M.S., 2000 Indian Institute of Technology Delhi, India

Thesis Advisor: Dr. Mukund Modak, Ph.D
Professor
Department of Biochemistry and Molecular Biology

Biochemistry and Molecular Biology
MSB E-609

Wednesday, October 29, 2008
3:00 p.m.


Abstract

The Klenow fragment of Escherichia coli DNA polymerase I houses catalytic centers for both polymerase and 3 5 exonuclease activities. 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 ~35 . Upon the incorporation of a mismatched nucleotide, the primer terminus is transferred from the polymerase site to the 3 5 exonuclease site designed for excision of the mismatched nucleotides. Thus, a movement of primer strand containing terminal mismatch would require 35 movement. This may further involve repositioning single-stranded template overhang to facilitate the movement. 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 821824. Since these residues are conserved in the A family DNA polymerases, this region was designated 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 229-fold, depending upon the site of mutation. The R821A and R822A/Y824A mutant enzymes showed maximum cleavage defect with single-stranded DNA (ssDNA), 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 coupled 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 non-substrate single-stranded template overhang, in a polarity-dependent manner. This binding then facilitates cleavage of the substrate at the exonuclease site.


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