The regulatory rep protein of adeno-associated virus binds to sequences within the c-H-ras promoter.
The large rep gene products (rep68 and rep78) of adeno-associated virus (AAV) are pleiotropic effector proteins which not only play a critical role in AAV DNA replication and in the trans-regulation of AAV promotor elements, but are also known for their onco-suppressive functions. We have previously demonstrated that the large AAV rep protein will strongly inhibit expression from the c-H-ras promoter, but not the murine osteosarcoma virus long terminal repeat (MSV-LTR) promoter. To investigate the possibility that rep may physically bind to these promoter sequences, specifically to GCTC motifs, we conducted electrophoretic mobility shift assays (EMSA) with a maltose binding protein-rep chimeric protein, MBP-rep68 delta, and synthetic double stranded DNA substrates of sequences selected from the c-H-ras and MSV-LTR promoters, as well as with the AAV TR. We find that MPB-rep68 delta bound the AAV TR DNA sequence (three motifs) most strongly, followed by the selected c-H-ras DNA sequence (two noninterfering motifs), and most poorly to the MSV-LTR DNA (one motif). These data are consistent with our previous study and suggest a direct mechanism of action for AAV rep inhibition of the c-H-ras promoter. Furthermore, the results suggest that the number of GCTC motifs, when closely associated, affect the affinity of rep binding. Finally, we find that MBP-rep68 delta also binds to the c-H-ras oligomer substrates which have secondary hairpin structures.
Batchu
R B
RB
Department of Obstetrics and Gynecology, University of Arkansas for Medical Science, Little Rock 72205.
Kotin
R M
RM
Hermonat
P L
PL
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D011905:Genes, ras; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2