The carboxyl terminus of the bacteriophage T4 DNA polymerase contacts its sliding clamp at the subunit interface.
The location of the interaction of the COOH terminus of the bacteriophage T4 DNA polymerase with its trimeric, circular sliding clamp has been established. A peptide corresponding to the COOH terminus of the DNA polymerase was labeled with a fluorophore and fluorescence spectroscopy used to show that it forms a specific complex with the sliding clamp by virtue of its low K(D) value (7.1 +/- 1.0 microM). The same peptide was labeled with a photoaffinity probe and cross-linked to the sliding clamp. Mass spectrometry of tryptic digests determined the sole linkage point to be Ala-159 on the sliding clamp, an amino acid that lies on the subunit interface. These results demonstrate that the COOH terminus of the DNA polymerase is inserted into the subunit interface of its sliding clamp, thereby conferring processivity to the DNA polymerase.
Alley
S C
SC
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Jones
A D
AD
Soumillion
P
P
Benkovic
S J
SJ
D017122:Bacteriophage T4; D003432:Cross-Linking Reagents; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005464:Fluorobenzenes; D008301:Maleimides; D008958:Models, Molecular; D015394:Molecular Structure; D009282:Naphthalenesulfonates; D010446:Peptide Fragments / Q000737:chemistry; D019920:Photoaffinity Labels; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D014764:Viral Proteins / Q000737:chemistry*
D003432:Cross-Linking Reagents; D005464:Fluorobenzenes; D008301:Maleimides; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C073711:N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide; D004259:DNA-Directed DNA Polymerase; D014357:Trypsin