Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase

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 ordi...

Full description

Saved in:
Bibliographic Details
Published in:Nucleic acids research 2007-01, Vol.35 (1), p.143-151
Main Authors: Hamilton, Michael D, Nuara, Anthony A, Gammon, Don B, Buller, R. Mark, Evans, David H
Format: Article
Language:English
Subjects:
Catalysis
DNA Repair
DNA Replication
DNA-Directed DNA Polymerase - metabolism
Kinetics
Models, Genetic
Nucleic Acid Enzymes
Oligodeoxyribonucleotides - metabolism
Poxvirus
Protein Binding
Recombination, Genetic
Surface Plasmon Resonance
Vaccinia virus
Citations: Items that cite this one
Online Access:Request full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkl1015