Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1.
In the presence of a single-stranded-DNA-binding protein (SSB), the elongation of primed DNA templates by DNA polymerase delta (pol delta) is dependent on ATP and two protein factors, activator 1 (A1) and proliferating cell nuclear antigen (PCNA). We have examined the interaction of these proteins with (dA)4500.(dT)12-18 by measuring their ability to form stable complexes with this DNA. In the presence of ATP, A1, PCNA, and pol delta formed a stable complex with DNA that could be isolated by gel filtration. Incubation of the isolated complex with dTTP resulted in the synthesis of poly(dT). While ATP was required for the formation of this complex, it was not required for the subsequent elongation of DNA. The temporal requirements for complex formation were determined. A1 was found to bind first, followed by the ATP-dependent addition of PCNA to the A1.DNA complex, while pol delta was added last. Each of these complexes could be isolated by gel filtration, indicating that they possessed a high degree of stability. The binding of PCNA to the A1-SSB-coated primed DNA occurred with adenosine 5'-[gamma-thio]triphosphate as well as ATP. However, the binding of pol delta to the PCNA.A1-DNA complex was observed only when the latter complex was formed in the presence of ATP. The complete complex was formed after incubation at 37 degrees C for 2 min, whereas no complex was detected after incubation at 0 degree C. These results indicate that these proteins act in a manner analogous to the accessory proteins that play critical roles in the elongation reaction catalyzed by T4 phage DNA polymerase and Escherichia coli DNA polymerase III.
Lee
S H
SH
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Hurwitz
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D004258:DNA Polymerase III; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D011067:Poly dA-dT / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D011067:Poly dA-dT; D000255:Adenosine Triphosphate; D004247:DNA; D011505:Protein-Tyrosine Kinases; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase