Determination of the origin-specific DNA-binding domain of polyomavirus large T antigen.
To map the DNA-binding domain of polyomavirus large T antigen, we constructed a set of plasmids coding for unidirectional carboxy- or amino-terminal deletion mutations in the large T antigen. Analysis of origin-specific DNA binding by mutant proteins expressed in Cos-1 cells revealed that the C-terminal boundary of the DNA-binding domain is at or near Glu-398. Fusion proteins of large T antigen lacking the first 200 N-terminal amino acids bound specifically to polyomavirus origin DNA; however, deletions beyond this site resulted in unstable proteins which could not be tested for DNA binding. Testing of point mutants and internal deletions by others suggested that the N-terminal boundary of the DNA-binding domain lies between amino acids 282 and 286. Taken together, these results locate the DNA-binding domain of polyomavirus large T antigen to the 116-amino-acid region between residues 282 and 398.
Sunstrom
N A
NA
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Acheson
N H
NH
Hassell
J A
JA
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D005822:Genetic Vectors; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011120:Polyomavirus / Q000235:genetics*; D015183:Restriction Mapping; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D008715:Methionine