Localized melting and structural changes in the SV40 origin of replication induced by T-antigen.
Replication of simian virus 40 (SV40) DNA is dependent upon the binding of the viral T-antigen to the SV40 origin of replication. Structural changes in the origin of replication induced by binding of T-antigen were probed by chemical modifications of the DNA. In the presence of ATP, T-antigen rendered two of three domains in the SV40 core origin hypersensitive to attack by either dimethyl sulfate or potassium permanganate (KMnO4). One of these domains, the early palindrome, was shown to contain an 8-bp region of melted DNA as determined from methylation of cytosine residues and by nuclease S1 cleavage of methylated DNA. DNA melting was not dependent upon either the hydrolysis of ATP or the binding of T-antigen to an adjacent site (site I). A second domain, the A/T element, was extensively modified by KMnO4 but no significant melting was detected. Rather, the pattern of modification indicates that T-antigen caused a conformational change of the double-stranded DNA in this region. These results suggest that T-antigen, in the presence of ATP, destabilizes the SV40 origin by melting and structurally deforming two flanking regions within the core origin sequence. These DNA structural changes may provide access to other replication factors, allowing complete denaturation of the SV40 origin and the initiation of SV40 DNA replication.
Borowiec
J A
JA
Graduate Program in Molecular Biology, Sloan-Kettering Cancer Center, New York, NY 10021.
Hurwitz
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology*; D001483:Base Sequence; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000502:physiology*; D004720:Endonucleases / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010084:Oxidation-Reduction; D011196:Potassium Permanganate; D012045:Regulatory Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D011196:Potassium Permanganate; D000255:Adenosine Triphosphate; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases