Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase

• 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
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkl1015

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.