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Nucleic Acids Research Advance Access published online on December 7, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl1015
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase

Michael D. Hamilton, Anthony A. Nuara1, Don B. Gammon, R. Mark Buller1 and David H. Evans*

Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry The University of Alberta, Edmonton, AB, Canada T6G 2H7 1 Department of Molecular Microbiology and Immunology, St Louis University Health Sciences Center 1402 South Grand Boulevard, St Louis, MO 63104, USA

*To whom correspondence should be addressed. Tel: +1 780 492 2308; Fax: +1 780 492 7521; Email: devans{at}ualberta.ca

Received August 29, 2006. Revised November 1, 2006. Accepted November 1, 2006.

Vaccinia virus DNA polymerase catalyzes duplex-by-duplex DNA joining reactions in vitro and many features of these recombination reactions are reprised in vivo. This can explain the intimate linkage between virus replication and genetic recombination. However, it is unclear why these apparently ordinary polymerases exhibit this unusual catalytic capacity. In this study, we have used different substrates to perform a detailed investigation of the mechanism of duplex-by-duplex recombination catalyzed by vaccinia DNA polymerase. When homologous, blunt-ended linear duplex substrates are incubated with vaccinia polymerase, in the presence of Mg2+ and dNTPs, the appearance of joint molecules is preceded by the exposure of complementary single-stranded sequences by the proofreading exonuclease. These intermediates anneal to form a population of joint molecules containing hybrid regions flanked by nicks, 1–5 nt gaps, and/or short overhangs. The products are relatively resistant to exonuclease (and polymerase) activity and thus accumulate in joining reactions. Surface plasmon resonance (SPR) measurements showed the enzyme has a relative binding affinity favoring blunt-ended duplexes over molecules bearing 3'-recessed gaps. Recombinant duplexes are the least favored ligands. These data suggest that a particular combination of otherwise ordinary enzymatic and DNA-binding properties, enable poxvirus DNA polymerases to promote duplex joining reactions.


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