Processive proofreading is intrinsic to T4 DNA polymerase

• Published in: The Journal of biological chemistry Vol. 267; no. 20; pp. 14157 - 14166
• Main Authors: REDDY, M. K, WEITZEL, S. E, VON HIPPEL, P. H
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-07-1992
• Subjects: Base Sequence; Biological and medical sciences; DNA Replication; DNA, Viral - biosynthesis; DNA, Viral - chemistry; DNA-Directed DNA Polymerase - isolation & purification; DNA-Directed DNA Polymerase - metabolism; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Heparin - pharmacology; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Oligodeoxyribonucleotides - chemical synthesis; phage T4; Replication; Substrate Specificity; T-Phages - enzymology; T-Phages - genetics; Templates, Genetic; Viral Proteins - isolation & purification; Viral Proteins - metabolism; DNA polymerase; Radiolabelling; Enzyme; Gel electrophoresis; Error; Myoviridae; Virus; T even phage; Exonuclease; Replication; Mechanism of action; Phage; Phage T4
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)49692-0

DNA replication occurs in vivo with very high processivity, meaning that the replication complex assembles at the origin(s)
of replication and then performs template-directed synthesis of DNA over virtually the entire genome without dissociation.
Such processivity also characterizes reconstituted replication holoenzyme complexes in vitro. However, the isolated DNA polymerases
are much less processive, especially under physiological conditions. In this paper we monitor the degree of processivity displayed
by the bacteriophage T4-coded DNA polymerase while in its proofreading mode by asking whether an isolated polymerase can "edit-out"
the 3'-terminal nucleotide from the primer (using the 3'---5'-exonuclease activity of the polymerase) and then switch into
the synthesis mode without dissociating from the DNA template. This "switch experiment" is accomplished by using mismatched
primer/template substrates as an experimental tool to mimic the situation that T4 DNA polymerase encounters after a misincorporation
event has occurred. By performing experiments under single-turnover conditions (obtained using a heparin trap), we demonstrate
that T4 DNA polymerase, upon encountering a misincorporated base, neither synthesizes the next base nor dissociates into solution.
Instead, with a greater than 80% probability, it removes the misincorporated base and then continues synthesis in a fully
processive manner. We also show that the removal of a doubly mispaired sequence from the 3'-terminus of the primer, followed
by synthesis, is comparably processive. In contrast, the apparent processivity of removing a triply mispaired terminus is
much reduced. Taken together, these observations are consistent with the notion that the "editing active site" of the T4 enzyme
optimally accommodates only two unpaired nucleotide residues. Our results do not support the idea that the exonuclease activity
of T4 DNA polymerase is highly selective for mismatched termini; they suggest instead that the dwell time at a misincorporated
base determines overall editing efficiency. The integrated results of this study provide additional insight into the structure
of the T4 DNA polymerase, as well as into the interactions between the polymerase and the polymerase accessory proteins that
are required to provide the holoenzyme complex with full processivity.