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Nucleic Acids Research 2005 33(7):2259-2268; doi:10.1093/nar/gki525
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Published online 20 April 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase

Michael D. Hamilton and David H. Evans*

Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, The University of Alberta Edmonton, AB, Canada T6G 2H7

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

Received January 10, 2005. Revised April 1, 2005. Accepted April 1, 2005.

Poxvirus DNA polymerases play a critical role in promoting virus recombination. To test if vaccinia polymerase (E9L) could mediate this effect by catalyzing the post-synaptic processing of recombinant joint molecules, we prepared substrates bearing a nick, a 3'-unpaired overhang, a 5' overhang, or both 3' and 5' overhangs. The sequence of the 5' overhang was also modified to permit or preclude branch migration across the joint site. These substrates were incubated with E9L, and the fate of the primer strand characterized under steady-state reaction conditions. E9L rapidly excises a mispaired 3' strand from a DNA duplex, producing a meta-stable nicked molecule that is a substrate for ligase. The reaction was not greatly affected by adding an unpaired 5' strand, but since such molecules cannot be processed into nicked intermediates, the 3'-ended strand continued to be subjected to exonucleolytic attack. Incorporating homology into the 5' overhang prevented this and permitted some strand assimilation, but such substrates also promoted strand-displacement DNA synthesis of a type predicted by the 1981 Moyer and Graves model for poxvirus replication. Single-strand annealing reactions are used by poxviruses to produce recombinant viruses and these data show that virus DNA polymerases can process DNA in such a manner as to both generate single-stranded substrates for such reactions and to facilitate the final processing of the reaction products.


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