Extrachromosomal recombination in vaccinia-infected cells requires a functional DNA polymerase participating at a level other than DNA replication.
Homologous recombination was measured in vaccinia-infected cells cotransfected with two plasmid recombination substrates. One plasmid contains a vaccinia protein lacZ coding region bearing a 1.1 kb 3' terminal deletion while the other plasmid contains a non-promoted lacZ coding region bearing a 1.1 kb 5' terminal deletion. Homologous recombination occurring between the 825 bp of lacZ common to both plasmids regenerates a functional lacZ gene from which B-galactosidase expression was measured. The entire 3 kb lacZ gene was used as a positive control. A panel of thermosensitive mutants was screened in cells either transfected with the positive control plasmid or cotransfected with the recombination substrates. A DNA - mutant, ts42, known to map to the viral DNA polymerase gene was found to be defective in recombination. Significantly, other DNA - mutants, ts17 or ts25, or other DNA polymerase mutants did not exhibit a defect in recombination similar to ts42. Inhibitors of viral DNA synthesis did not uniformly affect recombination. Cytosine arabinoside and aphidicolin inhibited B-galactosidase expression from the recombination substrates but not from the positive control plasmid, whereas hydroxyurea enhanced expression from both. Marker rescue with the cloned wildtype DNA polymerase gene repaired the defect in ts42. Southern and western analyses demonstrated that B-galactosidase activity was consistent with a recombined lacZ gene and unit size 116 kDa protein. Measurement of plasmid and viral DNA replication in cells infected with the different DNA - mutants indicated that recombination was independent of plasmid and viral DNA replication. Together these results suggest that the vaccinia DNA polymerase participates in homologous recombination at a level other than that of DNA replication.
Colinas
R J
RJ
Department of Microbiology and Immunology, Albany Medical College, New York 12208.
Condit
R C
RC
Paoletti
E
E
D000818:Animals; D002478:Cells, Cultured / Q000382:microbiology; D003561:Cytarabine / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D005819:Genetic Markers; D006358:Hot Temperature; D006918:Hydroxyurea / Q000494:pharmacology; D007763:Lac Operon; D009154:Mutation; D010746:Phosphonoacetic Acid / Q000494:pharmacology; D010957:Plasmids; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D014616:Vaccinia virus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014779:Virus Replication / Q000187:drug effects; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004279:DNA, Viral; D005819:Genetic Markers; D003561:Cytarabine; D004259:DNA-Directed DNA Polymerase; D001616:beta-Galactosidase; D010746:Phosphonoacetic Acid; D006918:Hydroxyurea