Analysis of the effects of charge cluster mutations in adeno-associated virus Rep68 protein in vitro

The Rep78 and Rep68 proteins of adeno-associated virus type 2 (AAV) are mul tifunctional proteins which are required for viral replication, regulation of AAV promoters, and preferential integration of the AAV genome into a reg ion of human chromosome 19. These proteins bind the hairpin structures form ed by the AAV inverted terminal repeat (ITR) origins of replication, make s ite- and strand-specific endonuclease cuts within the AAV ITRs, and display nucleoside triphosphate-dependent helicase activities. Additionally, sever al mutant Rep proteins display negative dominance in helicase and/or endonu clease assays when they are mixed with wild-type Rep78 or Rep68, suggesting that multimerization may be required for the helicase and endonuclease fun ctions. Using overlap extension PCR mutagenesis, we introduced mutations wi thin clusters of charged residues throughout the Rep68 moiety of a maltose binding protein-Rep68 fusion protein (MBP-Rep68 Delta) expressed in Escheri chia call cells. Several mutations disrupted the endonuclease and helicase activities; however, only one amino-terminal-charge cluster mutant protein (D40A-D42A-D44A) completely lost AAV hairpin DNA binding activity. Charge d uster mutations within two other regions abolished both endonuclease and he licase activities. One region contains a predicted alpha-helical structure (amino acids 371 to 393), and the other contains a putative 3,4 heptad repe at (coiled-coil) structure (amino acids 441 to 483). The defects displayed by these mutant proteins correlated with a weaker association with wild-typ e Rep68 protein, as measured in coimmunoprecipitation assays. These experim ents suggest that these regions of the Rep molecule are involved in Rep oli gomerization events critical for both helicase and endonuclease activities.