Baculovirus Alkaline Nuclease Possesses a 5′→3′ Exonuclease Activity and Associates with the DNA-Binding Protein LEF-3

Abstract

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 533 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 533 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␣) and DNA-binding protein (Red␤) of the Red-mediated homologous recombination system of bacteriophage .

The Herpes Simplex Virus Type 1 Alkaline Nuclease and Single-Stranded DNA Binding Protein Mediate Strand Exchange In Vitro

“…Consistent with this model, ICP8 interacts with UL12 (53,55). Interestingly, Mikhailov et al have reported that the baculovirus alkaline nuclease associates with the DNA binding protein LEF-3, suggesting that baculovirus may also encode a two-subunit recombinase (30).…”

Genomic and host range studies of Maruca vitrata nucleopolyhedrovirus

“…Several of these auxiliary genes have homologues in MaviNPV, including ptp-1, ubiquitin, p10, superoxide dismutases (sod), v-cath, chitinase, ecdysteroid UDPglucosyltransferase (egt), actin rearrangement-inducing factor-1 (arif-1), alkaline exonuclease (alk-exo) and pk-1. The only auxiliary gene conserved among all baculoviruses is alk-exo, which interacts with lef-3, possesses both endoand exonuclease activities and can degrade both single-and double-stranded DNA in a 59-39 direction (Mikhailov et al, 2003). Two additional auxiliary genes with unknown function, viral fibroblast growth factor (vfgf) and ubiquitin, were also considered conserved among all lepidopteran baculoviruses (Herniou et al, 2003); surprisingly, no homologue of vfgf gene was found in MaviNPV.…”

Identification of Domains in Autographa californica Multiple Nucleopolyhedrovirus Late Expression Factor 3 Required for Nuclear Transport of P143

“…LEF-3 also interacts with a viral alkaline nuclease (22), and it has been suggested that an alkaline nuclease-LEF-3 complex might be involved in repair and recombination of baculovirus genomes during replication. Because little is known about the mechanism of baculovirus replication, it will be of great interest to explore the functional role of other domains within LEF-3.…”