Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno-associated virus DNA replication.
The adeno-associated virus (AAV) terminal repeats (TR) are cis required, and the AAV encoded Rep78 protein is trans required, for AAV DNA replication. The Rep78 protein recognizes and interacts with at least three regions within the TR DNA. The major binding site, with the highest affinity for Rep78 binding, is within the TR stem (nt 36-16) and includes the 'core' GAGC trimer (GAGC3, nt 33-22; Fig. 2) sequence. In this study mutations were made within the GAGC trimer and these mutants assayed for their ability to allow for AAV double stranded (ds DNA, prepackaging DNA replication), and single stranded DNA (ss DNA, due to virion packaging) replication. Here, it is shown that when the two inside GAGC motifs are mutated, with only motif no. 1 left intact (see Fig. 2), the resulting AAV (mutA) genome was significantly defective for both ds DNA (17% of wild type) and ss DNA (9%). If the TRs contained only the two outside motifs intact (mutB), motifs no. 1 and 2, the AAV genome had a significant but reduced level of both ds (50%) and ss (34%) DNA replication. Finally, if only the middle motif no. 2 was mutated, with motifs no. 1 and 3 left intact (mutC), the resulting DNA replication for both ds and ss forms was essentially wild type (80% that of wild type). These data suggest that the GAGC trimer plays a role in AAV DNA replication, and that GAGC motif no. 3 is the most important of the three motifs for both ds and ss DNA replication.
Bishop
B M
BM
Department of Obstetrics, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Santin
A D
AD
Quirk
J G
JG
Hermonat
P L
PL
D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2