The T4 DNA polymerase accessory proteins form an ATP-dependent complex on a primer-template junction.
The DNA polymerase holoenzyme of bacteriophage T4 contains, besides the DNA polymerase itself (the gene 43 protein), a complex of the protein products of T4 genes 44 and 62 (a DNA-dependent ATPase) and of gene 45. Together, the 44/62 and 45 proteins form an ATP-dependent "sliding clamp" that holds a moving DNA polymerase molecule at the 3' terminus of a growing DNA chain. We have used a unique DNA fragment that forms a short hairpin helix with a single-stranded 5' tail (a "primer-template junction") to map the binding sites for these polymerase accessory proteins by DNA footprinting techniques. In the absence of the DNA polymerase, the accessory proteins protect from DNase I cleavage 19-20 nucleotides just behind the 3' end of the primer strand and 27-28 nucleotides on the complementary portion of the template strand. Detection of this DNA-protein complex requires the 44/62 and 45 proteins plus the nonhydrolyzable ATP analogue adenosine 5'-O-(thiotriphosphate). The complex is not detected in the presence of ATP. We suggest that ATP hydrolysis by the 44/62 protein normally activates the accessory proteins at a primer-template junction, permitting the DNA polymerase to bind and thus form the complete holoenzyme. However, when the polymerase is missing, as in these experiments, ATP hydrolysis is instead followed by a release (or loosening) of the accessory protein complex.
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
M M
MM
Munn
B M
BM
Alberts
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001345:Autoradiography; D001483:Base Sequence; D016172:DNA Fingerprinting; D004258:DNA Polymerase III; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology*; D013698:Templates, Genetic
D004279:DNA, Viral; D000255:Adenosine Triphosphate; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase