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Journal of Virology, February 2003, p. 2436-2444, Vol. 77, No. 4
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.4.2436-2444.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Baculovirus Alkaline Nuclease Possesses a 5'->3' Exonuclease Activity and Associates with the DNA-Binding Protein LEF-3{dagger}

Victor S. Mikhailov,1,2* Kazuhiro Okano,1 and George F. Rohrmann1*

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804,1 N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808, Russia2

Received 30 September 2002/ Accepted 12 November 2002

Alkaline nuclease (AN) of the Autographa californica multiple-capsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133) was expressed in recombinant baculovirus as a His6-tagged fusion and purified by sequential chromatography on Ni-NTA-agarose, DEAE-Toyopearl, and heparin-Sepharose. At all stages of purification, AcMNPV AN was found to copurify with a 44-kDa polypeptide which was identified as the baculovirus single-stranded DNA (ssDNA)-binding (SSB) protein, LEF-3. Sedimentation analysis in glycerol gradients of highly purified samples suggested that AN and LEF-3 are associated in a complex (designated *AN/L3), predominantly as heterodimers, although oligomeric forms containing both proteins were evident. In reactions with single- or double-stranded 62-mer oligonucleotides that were labeled with 32P at the 5' or 3' ends, *AN/L3 carried out exonucleolytic hydrolysis of both substrates exclusively in a 5'->3' direction. Saturation of ssDNA with an excess of LEF-3 prior to the addition of *AN/L3 resulted in a marked decrease in the rate of ssDNA hydrolysis. This suggests that excess LEF-3 may protect ssDNA from digestion by a AN-LEF-3 complex, thus regulating its activity in infected cells. The association of baculovirus AN with the viral SSB LEF-3 and the 5'->3' exonuclease activity of this complex suggests that AN and LEF-3 may participate in homologous recombination of the baculovirus genome in a manner similar to that of exonuclease (Red{alpha}) and DNA-binding protein (Redß) of the Red-mediated homologous recombination system of bacteriophage {lambda}.

* Corresponding author. Mailing address: Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804. Fax: (541) 737-0496. Phone for George F. Rohrmann: (541) 737-1793. E-mail: rohrmanng{at}orst.edu. Phone for Victor S. Mikhailov: (541) 737-1794. E-mail: Victor.Mikhailov@orst.edu.

{dagger} This is Technical Report no. 11932 from the Oregon State University Agricultural Experiment Station.

Journal of Virology, February 2003, p. 2436-2444, Vol. 77, No. 4
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.4.2436-2444.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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