Baculovirus alkaline nuclease possesses a 5'-->3' exonuclease activity and associates with the DNA-binding protein LEF-3.
Alkaline nuclease (AN) of the Autographa californica multiple-capsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133) was expressed in recombinant baculovirus as a His(6)-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 (32)P 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 (Redalpha) and DNA-binding protein (Redbeta) of the Red-mediated homologous recombination system of bacteriophage lambda.
Mikhailov
Victor S
VS
Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA. Victor.Mikhailov@orst.edu
Okano
Kazuhiro
K
Rohrmann
George F
GF
D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000201:enzymology*; D009841:Oligonucleotides / Q000378:metabolism; D011995:Recombination, Genetic; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera; D014764:Viral Proteins / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D014764:Viral Proteins; C094647:late expression factor 3, baculovirus; D005092:Exonucleases; D012260:Ribonucleases; C066964:viral alkaline nuclease