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 Esch erichia coli as a fusion protein with maltose-binding protein (MBP-Rep 68 Delta) has previously been shown to possess DNA-DNA helicase activi ty, as does the purified wild-type Rep68. In the present study, we dem onstrate that MBP-Rep68 Delta also catalyzes the unwinding of a DNA-RN A 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 MB P-Rep68 Delta was comparable to its DNA-DNA helicase activity. However , MBP-Rep68 Delta lacked the ability to unwind a blunt-ended DNA-RNA s ubstrate and RNA-RNA duplexes. We have also demonstrated that MBP-Rep6 8 Delta has ATPase activity which is enhanced by the presence of singl e-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 lack s DNA-RNA helicase and ATPase activities but also inhibits the helicas e activity of MBP-Rep68 Delta DNA-RNA helicase activity of Rep protein s might play a pivotal role in the regulation of AAV gene expression b y AAV Rep proteins.