HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 26, 17979-17990, June 27, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/26/17979    most recent M801053200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Kukreti, P. Articles by Modak, M. J. PubMed PubMed Citation Articles by Kukreti, P. Articles by Modak, M. J. Social Bookmarking                      What's this?

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

THE RRRY MOTIF IS NECESSARY FOR THE BINDING OF SINGLE-STRANDED DNA SUBSTRATE AND THE TEMPLATE STRAND OF THE MISMATCHED DUPLEX^*

From the Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, New Jersey 07103

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.

Received for publication, February 8, 2008 , and in revised form, April 24, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM 36307 (NIGMS) and AI-064477 (NIAID). A part of this work was presented at the annual meeting of FASEB (29). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Biochemistry and Molecular Biology, UMD-New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103. Tel.: 973-972-4053; Fax: 973-972-5594; E-mail: modak{at}umdnj.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?