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Journal of Virology, March 2002, p. 2287-2297, Vol. 76, No. 5
0022-538X/02/$04.00+0     DOI: 10.1128/jvi.76.5.2287-2297.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Baculovirus Replication Factor LEF-1 Is a DNA Primase{dagger}

*** Victor S. Mikhailov1,2* 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 29 August 2001/ Accepted 7 November 2001

The baculovirus replication factors LEF-1 and LEF-2 of the Autographa californica multinucleocapsid nucleopolyhedrovirus were overexpressed as fusions containing a hemagglutinin (HA) epitope and a HIS6 tag using recombinant baculoviruses. LEF-1 was purified to near homogeneity and found to have primase activity in an indirect assay employing Escherichia coli DNA polymerase I (Klenow enzyme) and poly(dT) template. The LEF-1 primase products were also directly characterized by electrophoresis in 20% polyacrylamide-8 M urea gels and agarose gels. Primer synthesis was time dependent, and products of several hundred nucleotides or more were observed from the M13 single-stranded DNA (ssDNA) template. The LEF-1 primase was absolutely dependent on divalent cations (Mg2+), and optimal activity was supported by 10 mM MgCl2. An alkaline pH (8.8 to 9.4) was optimal, whereas monovalent salt (KCl) was inhibitory. Mutation of an invariant aspartic acid in a putative primase domain caused LEF-1 activity to be abolished. Upon ultracentrifugation in glycerol gradients, LEF-1 was found to have a sedimentation coefficient of 3S that is consistent with its being present as a monomer. Elution profiles of LEF-1 and LEF-2 from ssDNA-cellulose and DEAE resin suggested that LEF-2 may bind to both DNA and LEF-1.

* Corresponding author. Mailing address: N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Vavilov St. 26, Moscow 117808, Russia. Phone: (7-095)1358847. Fax: (7-095)1358012. E-mail: vmikhailov{at}proxima.idb.ac.ru.

{dagger} Technical report no. 11820 from the Oregon State University Agricultural Experiment Station.

Journal of Virology, March 2002, p. 2287-2297, Vol. 76, No. 5
0022-538X/02/$04.00+0     DOI: 10.1128/jvi.76.5.2287-2297.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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