Dissociation of bacteriophage T4 DNA polymerase and its processivity clamp after completion of Okazaki fragment synthesis.
The mechanism of bacteriophage T4 DNA polymerase (gp43) and clamp (gp45) protein dissociation from the holoenzyme DNA complex was investigated under conditions simulating the environment encountered upon completion of an Okazaki fragment. Lagging strand DNA synthesis was approximated using a synthetic construct comprised of a doubly biotinylated, streptavidin-bound 62-mer DNA template, paired with complementary primers to generate an internal 12-base gap where the 5'-end primer contained either a 5'-OH (DNA primer) or a 5'-triphosphate (RNA primer) group. Rapid kinetic measurements revealed that upon encountering the blocking primer, the holoenzyme either dissociates from DNA (approximately 40%) or strand-displaces the blocking strand (approximately 60%). The two blocking oligonucleotides (DNA or RNA) induce a 30-50-fold increase in the rate of holoenzyme dissociation, with both polymerase and clamp proteins dissociating simultaneously. Inhibition of ATP hydrolysis by ATP-gamma-S did not have a measurable effect upon holoenzyme dissociation from DNA. The presence of gp32, the single-strand binding protein, caused a small (3-fold) increase in the rate constant for dissociation.
Carver
T E
TE
Department of Chemistry, 152 Davey Laboratory, Pennsylvania State University, University Park 16802, USA.
Sexton
D J
DJ
Benkovic
S J
SJ
D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry*; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D004279:DNA, Viral; C022262:Okazaki fragments; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004247:DNA; D004259:DNA-Directed DNA Polymerase