The DNA-binding properties of polyomavirus large T antigen are altered by ATP and other nucleotides.
We have examined the influence of ATP on the DNA-binding properties of polyomavirus large T antigen (Py TAg). Utilizing nitrocellulose filter binding, DNase I footprinting, and gel mobility shift assays, we observed that ATP increased Py TAg binding to DNA fragments containing either all Py TAg-binding sites (whole origin) or those sites within (core origin) or adjacent to (early) the origin of replication. Even nonspecific binding to DNA fragments lacking Py TAg-binding sites was increased somewhat by ATP. Binding to the core origin was increased to a greater extent than binding to other DNA fragments tested. Gel band mobility shift assays revealed that ATP increased the production of core origin-specific Py TAg-DNA complexes of high molecular weight. ATP stimulation depended on the presence of MgCl2. Other nucleotides and nonhydrolyzable ATP analogs also increased Py TAg binding to the core origin but to various degrees: ATP, dATP, 5'-adenylyl imidodiphosphate (AMPPNP) greater than 5'-adenylyl methylenediphosphate (AMPPCP) greater than dCTP greater than UTP greater than TTP. GTP and dGTP did not increase DNA binding by Py TAg. The rates of association and disassociation of Py TAg with all the DNA fragments were altered by the presence of ATP. DNase I footprinting showed that ATP extensively extended the region protected within the core origin and also produced a distinctive DNase I-hypersensitive site on the late strand at nucleotides 5255 to 5262 (TTACTATG).
Lorimer
H E
HE
Department of Biological Sciences, Columbia University, New York, New York 10027.
Wang
E H
EH
Prives
C
C
D000227:Adenine Nucleotides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D007700:Kinetics; D009710:Nucleotide Mapping; D011120:Polyomavirus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology*
D000227:Adenine Nucleotides; D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012265:Ribonucleotides; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I