Recombination in vitro between herpes simplex virus type 1 a sequences.
We have partially purified an activity from extracts of cells infected with herpes simplex virus type 1 that mediates recombination between repeated copies of the 317-base-pair a sequence of herpes simplex virus type 1. Recombination leads to deletion of a lacZ indicator gene situated between two directly repeated copies of the a sequence and is scored by transformation of lacZ- Escherichia coli. The two products of the reaction can be observed directly by restriction enzyme digestion and Southern blot analysis. The recombinase activity is also detectable, but at a lower level, in uninfected cell extracts. The DNA substrate must contain the two a sequences arranged in direct orientation to generate the lacZ deletion. However, when the a sequences are arranged in inverted orientation, an inversion results. A substrate with two homologous sequences of size and G + C content similar to the a sequence undergoes recombination at a much lower frequency. The reaction requires a divalent cation (Mg2+ or Mn2+) but not ATP or any other nucleoside triphosphate. The simple requirements and specificity for the a sequence suggest that the recombination may proceed by a site-specific mechanism.
Bruckner
R C
RC
Department of Biochemistry, Stanford University School of Medicine, CA 94305.
Dutch
R E
RE
Zemelman
B V
BV
Mocarski
E S
ES
Lehman
I R
IR
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D019426:Integrases; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009994:Osmolar Concentration; D045522:Recombinases; D011995:Recombination, Genetic; D015183:Restriction Mapping; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004279:DNA, Viral; D045522:Recombinases; D008345:Manganese; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1; D001616:beta-Galactosidase; D008274:Magnesium