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 multifunctional proteins which are required for viral replication, regulation of AAV promoters, and preferential integration of the AAV genome into a region of human chromosome 19. These proteins bind the hairpin structures formed by the AAV inverted terminal repeat (ITR) origins of replication, make site- and strand-specific endonuclease cuts within the AAV ITRs, and display nucleoside triphosphate-dependent helicase activities. Additionally, several mutant Rep proteins display negative dominance in helicase and/or endonuclease assays when they are mixed with wild-type Rep78 or Rep68, suggesting that multimerization may be required for the helicase and endonuclease functions. Using overlap extension PCR mutagenesis, we introduced mutations within clusters of charged residues throughout the Rep68 moiety of a maltose binding protein-Rep68 fusion protein (MBP-Rep68Delta) expressed in Escherichia coli 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 cluster mutations within two other regions abolished both endonuclease and helicase activities. One region contains a predicted alpha-helical structure (amino acids 371 to 393), and the other contains a putative 3,4 heptad repeat (coiled-coil) structure (amino acids 441 to 483). The defects displayed by these mutant proteins correlated with a weaker association with wild-type Rep68 protein, as measured in coimmunoprecipitation assays. These experiments suggest that these regions of the Rep molecule are involved in Rep oligomerization events critical for both helicase and endonuclease activities.
Davis
M D
MD
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.
Wonderling
R S
RS
Walker
S L
SL
Owens
R A
RA
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology; D000229:Dependovirus / Q000737:chemistry*; D004720:Endonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology