IDENTIFICATION OF MUTANT ADENOASSOCIATED VIRUS REP PROTEINS WHICH AREDOMINANT-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 durin g AAV DNA replication. Previous studies showed that AAV Rep78 and Rep6 8 could unwind a DNA partial duplex of 26 bp. In this work it is demon strated that nuclear extracts of human 293 cells containing wild-type Rep68 can unwind partial DNA duplexes up to 160 bp long. Mutant Rep pr oteins with either a histidine substituted for lysine 340 or a deletio n 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.