An adeno-associated virus (AAV) initiator protein, Rep78, catalyzes the cleavage and ligation of single-stranded AAV ori DNA.
The Rep78 protein of adeno-associated virus (AAV) contains amino acid sequence motifs common to rolling-circle replication (RCR) initiator proteins. In this report, we describe RCR initiator-like activities of Rep78. We demonstrate that a maltose-binding protein (MBP)-Rep78 fusion protein can catalyze the cleavage and ligation of single-stranded DNA substrates derived from the AAV origin of replication. Rep-mediated single-stranded DNA cleavage was strictly dependent on the presence of certain divalent cations (e.g., Mn(2+) or Mg(2+)) but did not require the presence of a nucleoside triphosphate cofactor. Electrophoretic mobility shift assays demonstrated that binding of single-stranded DNA by MBP-Rep78 was influenced by the length of the substrate as well as the presence of potential single-stranded cis-acting sequence elements. Site-directed mutagenesis was used to examine the role of specific tyrosine residues within a conserved RCR motif (motif 3) of Rep78. Replacement of Tyr-156 with phenylalanine abolished the ability of MBP-Rep78 to mediate the cleavage and ligation of single-stranded DNA substrates but not the ability to stably bind single-stranded DNA. The cleaving-joining activity of Rep78 is consistent with the mechanism of replicative intermediate dimer resolution proposed for the autonomous parvoviruses and may have implications for targeted integration of recombinant AAV vectors.
Smith
R H
RH
Laboratory of Molecular Hematology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
Kotin
R M
RM
D001483:Base Sequence; D002384:Catalysis; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018741:Replication Origin / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2