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J. Biol. Chem., Vol. 282, Issue 2, 1432-1444, January 12, 2007

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Structural and Biochemical Investigation of the Role in Proofreading of a Hairpin Loop Found in the Exonuclease Domain of a Replicative DNA Polymerase of the B Family^*

Matthew Hogg¹, Pierre Aller¹, William Konigsberg, Susan S. Wallace², and Sylvie Doublié³

From the Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405 and the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510-3219

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4Å crystal structure of the hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the hairpin loop.

Received for publication, June 13, 2006 , and in revised form, November 9, 2006.

The atomic coordinates and structure factors (code 2DTU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by National Institutes of Health Grant CA52040 (to S. S. W.). 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 Both authors contributed equally to this work.

³ Recipient of funding from the Human Frontier Science Program Organization. To whom correspondence may be addressed. Tel.: 802-656-9531; Fax: 802-656-8749; E-mail: sdoublie{at}uvm.edu.

² To whom correspondence may be addressed: Dept. of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Dr., Burlington, VT 05405-0068. Tel.: 802-656-2164; Fax: 802-656-8749; E-mail: swallace{at}uvm.edu.

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