Strand-specific recognition of a synthetic DNA replication fork by the SV40 large tumor antigen.
The mechanism by which DNA helicases unwind DNA was tested; an "unwinding complex" between the SV40 large tumor antigen (T antigen) and a DNA molecule designed to resemble a replication fork was probed. In an adenosine triphosphate (ATP)-dependent reaction, T antigen quantitatively recognized this synthetic replication fork and bound the DNA primarily as a hexamer. The T antigen bound only one of the two strands at the fork, an asymmetric interaction consistent with the 3'----5' directionality of the DNA helicase activity of T antigen. Binding to chemically modified DNA substrates indicated that the DNA helicase recognized the DNA primarily through the sugar-phosphate backbone. Ethylation of six top strand phosphates at the junction of single-stranded and double-stranded DNA inhibited the DNA helicase activity of T antigen. Neither a 3' single-stranded end on the DNA substrate nor ATP hydrolysis was required for T antigen to bind the replication fork. These data suggest that T antigen can directly bind the replication fork through recognition of a fork-specific structure.
SenGupta
D J
DJ
Department of Biochemistry, New York University Medical Center, NY 10016.
Borowiec
J A
JA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology*; D004265:DNA Helicases / Q000502:physiology; D004261:DNA Replication / Q000276:immunology*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005038:Ethylnitrosourea / Q000494:pharmacology; D005561:Formates / Q000494:pharmacology; D011196:Potassium Permanganate / Q000494:pharmacology; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D013997:Time Factors
D000952:Antigens, Polyomavirus Transforming; D004277:DNA, Single-Stranded; D005561:Formates; D013463:Sulfuric Acid Esters; D011196:Potassium Permanganate; C030544:formic acid; D000255:Adenosine Triphosphate; D004265:DNA Helicases; C007482:dimethyl sulfate; C011612:diethyl sulfate; D004047:Diethyl Pyrocarbonate; D005038:Ethylnitrosourea