Identification of mutant adeno-associated virus Rep proteins which are dominant-negative for DNA helicase activity.
Adeno-associated virus type 2 (AAV) Rep proteins have been postulated to play a role in unwinding the 145-bp inverted terminal repeats during AAV DNA replication. Previous studies showed that AAV Rep78 and Rep68 could unwind a DNA partial duplex of 26 bp. In this work it is demonstrated that nuclear extracts of human 293 cells containing wild-type Rep68 can unwind partial DNA duplexes up to 160 bp long. Mutant Rep proteins with either a histidine substituted for lysine 340 or a deletion of methionine 225 had no detectable helicase activity and inhibited the helicase activity of wild-type Rep68 protein. This observation is consistent with the model that the functional form of the Rep proteins is a multimer.
Kyöstiö
S R
SR
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA, 20892.
Owens
R A
RA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology; D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000229:Dependovirus / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D016325:HIV Long Terminal Repeat; D006801:Humans; D015636:Magnesium Chloride / Q000494:pharmacology; D009154:Mutation; D010957:Plasmids; D011993:Recombinant Fusion Proteins; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015636:Magnesium Chloride; C068734:rep proteins, Adeno-associated virus 2; D000255:Adenosine Triphosphate; D004265:DNA Helicases