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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
5
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pubmed:dateCreated |
2002-2-11
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pubmed:abstractText |
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 HIS(6) 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 (Mg(2+)), and optimal activity was supported by 10 mM MgCl(2). 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.
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pubmed:grant |
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pubmed:commentsCorrections |
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Mar
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pubmed:issn |
0022-538X
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
76
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
2287-97
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pubmed:dateRevised |
2009-11-18
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pubmed:meshHeading |
pubmed-meshheading:11836407-Amino Acid Sequence,
pubmed-meshheading:11836407-Animals,
pubmed-meshheading:11836407-Baculoviridae,
pubmed-meshheading:11836407-Base Sequence,
pubmed-meshheading:11836407-Cells, Cultured,
pubmed-meshheading:11836407-DNA Primase,
pubmed-meshheading:11836407-Molecular Sequence Data,
pubmed-meshheading:11836407-Plasmids,
pubmed-meshheading:11836407-Recombination, Genetic,
pubmed-meshheading:11836407-Sequence Alignment,
pubmed-meshheading:11836407-Sequence Analysis, DNA,
pubmed-meshheading:11836407-Spodoptera,
pubmed-meshheading:11836407-Viral Proteins
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pubmed:year |
2002
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pubmed:articleTitle |
Baculovirus replication factor LEF-1 is a DNA primase.
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pubmed:affiliation |
Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804, USA. vmikhailov@proxima.idb.ac.ru
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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