A MALTOSE-BINDING PROTEIN ADENOASSOCIATED 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.