A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities.
The adeno-associated virus type 2 (AAV) Rep68 protein produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP-Rep68 delta) has previously been shown to possess DNA-DNA helicase activity, as does the purified wild-type Rep68. In the present study, we demonstrate that MBP-Rep68 delta also catalyzes the unwinding of a DNA-RNA hybrid. MBP-Rep68 delta-mediated DNA-RNA helicase activity required ATP hydrolysis and the presence of Mg2+ ions and was inhibited by high ionic strength. The efficiency of the DNA-RNA helicase activity of MBP-Rep68 delta was comparable to its DNA-DNA helicase activity. However, MBP-Rep68 delta lacked the ability to unwind a blunt-ended DNA-RNA substrate and RNA-RNA duplexes. We have also demonstrated that MBP-Rep68 delta has ATPase activity which is enhanced by the presence of single-stranded DNA but not by RNA. The MBP-Rep68 delta NTP mutant protein, which has a lysine-to-histidine substitution at amino acid 340 in the putative nucleoside triphosphate-binding site of Rep68, not only lacks DNA-RNA helicase and ATPase activities but also inhibits the helicase activity of MBP-Rep68 delta. DNA-RNA helicase activity of Rep proteins might play a pivotal role in the regulation of AAV gene expression by AAV Rep proteins.
Wonderling
R S
RS
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.
Kyöstiö
S R
SR
Owens
R A
RA
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009841:Oligonucleotides; D012313:RNA / Q000378:metabolism; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C461599:maltose transport system, E coli; C068734:rep proteins, Adeno-associated virus 2; D012313:RNA; D008320:Maltose; D004247:DNA; D012316:RNA Nucleotidyltransferases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D020365:RNA Helicases