Nonspecific DNA binding activity of simian virus 40 large T antigen: evidence for the cooperation of two regions for full activity.
We generated a series of COOH-terminal truncated simian virus 40 large tumor (T) antigens by using oligonucleotide-directed site-specific mutagenesis. The mutant proteins [T(1-650) to T(1-516)] were expressed in insect cells infected with recombinant baculoviruses. T(1-623) and shorter proteins [T(1-621) to T(1-516)] appeared to be structurally changed in a region between residues 269 and 522, as determined by increased sensitivities to trypsin digestion and by altered reactivities to several monoclonal antibodies. These same mutant proteins bound significantly less nonorigin plasmid DNA (15%) and calf thymus DNA (25%) than longer proteins [T(1-625) to T(1-708)]. However, all mutant T antigens exhibited a nearly wild-type level of viral origin-specific DNA binding and binding to a helicase substrate DNA. This indicated that binding to origin and helicase substrate DNAs is separable from about 85% of nonspecific binding to double-stranded DNA. As an independent confirmation that a region distinct from the origin-binding domain (amino acids 147 to 247) is involved in nonspecific DNA binding, we found that up to 96% of this latter activity was specifically inhibited in wild-type T antigen by several monoclonal antibodies which collectively bind to the region between residues 269 and 522. In order to investigate the relationship between the origin-binding domain and the second region, we performed origin-specific DNA binding assays with increasing amounts of calf thymus DNA as competitor. The results suggest that this second region is not an independent nonspecific DNA binding domain. Rather, it most likely cooperates with the origin-binding domain to give rise to wild-type levels of nonspecific DNA binding. Our results further suggest that most of the nonspecific binding to double-stranded DNA is involved in a function other than direct recognition and binding to the pentanucleotides at the replication origin on simian virus 40 DNA.
Lin
H J
HJ
School of Life and Health Sciences, University of Delaware, Newark 19716.
Upson
R H
RH
Simmons
D T
DT
D000818:Animals; D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014779:Virus Replication
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D004247:DNA; D010450:Endopeptidases; D004265:DNA Helicases