Two types of replication proteins increase the rate at which T4 DNA polymerase traverses the helical regions in a single-stranded DNA template.

We have recently developed an in vitro DNA synthesis system in which a synthetic heptaribonucleotide pairs with a unique site on a single-stranded fd DNA molecule and thereby primes the growth of new DNA strands from this single point (Huang, C.-C., and Hearst, J. E. (1980) Anal. Biochem. 103, 127-139). In this report, we use this system to investigate the mechanism by which various bacteriophage T4 DNA replication proteins stimulate the T4 DNA polymerase. We find that with the "polymerase accessory proteins" present (the T4 gene 44/62 and 45 proteins), the DNA polymerase proceeds rather rapidly through the occasional hairpin helices which otherwise interrupt the progress of this enzyme along single-stranded DNA templates. By using a potent inhibitor of the 44/62 ATPase, ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)), we have obtained data which suggest that ATP hydrolysis is required for the formation of a polymerase accessory protein-DNA template complex, and that this complex then persists, serving as a sliding clamp which greatly increases the strength of binding between a T4 DNA polymerase molecule and its 3'OH primer template end. The progress of the T4 DNA polymerase though hairpin helices in the DNA template is also stimulated by addition of the T4 helix-destabilizing protein (gene 32 protein). The effect of the 44/62 and 45 proteins is independent of the effect of the 32 protein in this assay, and the rate of polymerase travel over the strongest hairpin helices is increased more than 40-fold in the presence of these four additional proteins.

• Publication year

  1981

• Venue

  Journal of Biological Chemistry

• Publication date

  1981-04-25

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1016/s0021-9258(19)69569-4PMID 6971292
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• The effects of the 44/62 and 45 proteins are independent of the 32 protein, and the combined presence of these proteins increases traversal of the strongest hairpin helices by more than 40-fold.

  Confidence 0.91

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• T4 gene 32 protein also stimulates T4 DNA polymerase as it moves through hairpin helices in the DNA template.

  Confidence 0.93

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• ATP hydrolysis by the 44/62 ATPase is required for forming a polymerase accessory protein-DNA template complex that acts as a sliding clamp and strengthens T4 DNA polymerase binding to the primer-template end.

  Confidence 0.95

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• Polymerase accessory proteins let T4 DNA polymerase traverse occasional hairpin helices in single-stranded DNA more rapidly.

  Confidence 0.97

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review

• 44/62 atpase

  enzyme, protein

  The ATP-hydrolyzing activity associated with the T4 gene 44/62 proteins.

  Aliases: gene 44/62 ATPase, 44/62 protein ATPase

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• atp gamma s

  molecule, inhibitor

  A nonhydrolyzable ATP analog used here as an inhibitor of the 44/62 ATPase.

  Aliases: adenosine 5'-O-(3-thiotriphosphate), ATPγS

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• hairpin helices

  nucleic acid structure, phenomenon

  Secondary-structure elements in the single-stranded DNA template that can impede polymerase movement.

  Aliases: hairpins, helical regions

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• in vitro dna synthesis system

  method

  An experimental setup using a synthetic primer on single-stranded fd DNA to study DNA synthesis in a test tube.

  Aliases: DNA synthesis system

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• polymerase accessory proteins

  protein complex, protein

  The T4 gene 44/62 and 45 proteins that associate with the polymerase during template synthesis.

  Aliases: 44/62 and 45 proteins, gene 44/62 and 45 proteins

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• single-stranded fd dna template

  nucleic acid, template

  A single-stranded fd bacteriophage DNA molecule that serves as the template for DNA polymerase extension.

  Aliases: ss fd DNA, fd DNA template

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• synthetic heptaribonucleotide

  oligonucleotide, primer

  A synthetic seven-ribonucleotide oligonucleotide used as the primer in the DNA synthesis assay.

  Aliases: heptaribonucleotide primer

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• t4 dna polymerase

  enzyme, protein

  The bacteriophage T4 DNA polymerase enzyme whose movement along the template is being studied.

  Aliases: T4 polymerase

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review
• t4 gene 32 protein

  protein

  The T4 helix-destabilizing single-stranded DNA-binding protein used in the replication assay.

  Aliases: gene 32 protein, gp32

  박진우 (dztg5apj7m) extractionB (s683577b42) reviewAnonymous (12632b8b5f) reviewjihoonc (k5vuy3tzcm) review

• Pauses at positions of secondary structure during in vitro replication of single-stranded fd bacteriophage DNA by T4 DNA polymerase.

  1980cited by this paper
• Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system.

  1980cited by this paper
• Processiveness of DNA polymerases. A comparative study using a simple procedure.

  1979cited by this paper
• Purification of the gene 43, 44, 45, and 62 proteins of the bacteriophage T4 DNA replication apparatus.

  1979cited by this paper
• Purification of the T4 gene 32 protein free from detectable deoxyribonuclease activities.

  1979cited by this paper
• The effect of template secondary structure on vaccinia DNA polymerase.

  1979cited by this paper
• DNA replication by bacteriophage T4 proteins: role of the DNA-delay gene 61 in the chain-initiation reaction.

  1979cited by this paper
• Probing DNA replication mechanisms with the T4 bacteriophage in vitro system.

  1979cited by this paper
• DNA replication by bacteriophage T4 proteins. The T4 43, 32, 44--62, And 45 proteins are required for strand displacement synthesis at nicks in duplex DNA.

  1979cited by this paper
• An ATP stimulation of T4 DNA polymerase mediated via T4 gene 44/62 and 45 proteins. The requirement for ATP hydrolysis.

  1978cited by this paper
• Nucleotide sequence of bacteriophage fd DNA.

  1978cited by this paper
• Analysis of a T4 DNA replication protein complex. Studies of the DNA recognition site for T4 gene 44/62 and 45 protein-catalyzed ATP hydrolysis.

  1978cited by this paper
• Structure of the orgin of DNA replication of bacteriophage fd.

  1978cited by this paper
• Enzymatic unwinding of DNA. III. Mode of action of Escherichia coli DNA unwinding enzyme.

  1977cited by this paper
• A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication.

  1977cited by this paper
• Recent excitement in the DNA replication problem

  1977cited by this paper
• Replication of duplex DNA by bacteriophage T7 DNA polymerase and gene 4 protein is accompanied by hydrolysis of nucleoside 5'-triphosphates.

  1977cited by this paper
• Studies on the mechanism of enzymatic DNA elongation by Escherichia coli DNA polymerase II.

  1976cited by this paper
• Psoralen-crosslinked secondary structure map of single-stranded virus DNA.

  1976cited by this paper
• Mechanism of DNA elongation catalyzed by Escherichia coli DNA polymerase III, dnaZ protein, and DNA elongation factors I and III.

  1976cited by this paper
• Reconstruction of bacteriophage T4 DNA replication apparatus from purified components: rolling circle replication following de novo chain initiation on a single-stranded circular DNA template.

  1975cited by this paper
• In vitro complementation as an assay for new proteins required for bacteriophage T4 DNA replication: purification of the complex specified by T4 genes 44 and 62.

  1972cited by this paper
• T4 Bacteriophage Gene 32: A Structural Protein in the Replication and Recombination of DNA

  1970cited by this paper
• DNA replication: the rolling circle model.

  1968cited by this paper
• The structural gene for deoxyribonucleic acid polymerase in bacteriophages T4 and T5.

  1965cited by this paper

• The bacteriophage T4 replisome: a model system for understanding DNA replication mechanisms

  2025cites this paper
• Life at the replication fork: A scientific and personal journey

  2024cites this paper
• C-terminal domain of T4 gene 32 protein enables rapid filament reorganization and dissociation.

  2024cites this paper
• Dynamic structure of T4 gene 32 protein filaments facilitates rapid noncooperative protein dissociation

  2023cites this paper
• From Processivity to Genome Maintenance: The Many Roles of Sliding Clamps

  2022cites this paper
• Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch

  2020cites this paper
• Structure of the human clamp loader bound to the sliding clamp: a further twist on AAA+ mechanism

  2020cites this paper
• Proteinirotein interactions at a DNA replication fork : bacteriophag T 4 as a-model

  2020cites this paper
• Understanding DNA replication by the bacteriophage T4 replisome

  2017cites this paper
• DNA Replication by the Bacteriophage T 4 Replisome 1 Understanding DNA Replication by the Bacteriophage T 4 Replisome

  2017cites this paper
• Mismatch repair in Gram-positive bacteria.

  2016cites this paper
• Molecular Dissection of the Homotrimeric Sliding Clamp of T4 Phage: Two Domains of a Subunit Display Asymmetric Characteristics.

  2016cites this paper
• Structural Mechanisms of the Sliding Clamp and Sliding Clamp Loader: Insights into Disease and Function: A Dissertation

  2016cites this paper
• Dynamics of Open DNA Sliding Clamps

  2016cites this paper
• Coordinated DNA Replication by the Bacteriophage T4 Replisome

  2015cites this paper
• Anti-cancer implications of small molecule compounds targeting proliferating cell nuclear antigen

  2014cites this paper
• Engineering processive DNA polymerases with maximum benefit at minimum cost

  2014cites this paper
• DnaN clamp zones provide a platform for spatiotemporal coupling of mismatch detection to DNA replication

  2013cites this paper
• Studies of DNA-Replication at the Single Molecule Level Using Magnetic Tweezers

  2011cites this paper
• Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome

  2009cites this paper
• Motors, switches, and contacts in the replisome.

  2009cites this paper
• Repetitive lagging strand DNA synthesis by the bacteriophage T4 replisome.

  2008cites this paper
• Multiple ATP Binding Is Required to Stabilize the “Activated” (Clamp Open) Clamp Loader of the T4 DNA Replication Complex*

  2008cites this paper
• Thermodynamics and kinetics of DNA-protein interactions from single molecule force spectroscopy

  2008cites this paper
• Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion

  2008cites this paper
• The control mechanism for lagging strand polymerase recycling during bacteriophage T4 DNA replication.

  2006cites this paper
• Bayesian shadows of molecular mechanisms cast in the light of evolution.

  2006cites this paper
• Replication Hits 50

  2005cites this paper
• Evolutionary clues to eukaryotic DNA clamp-loading mechanisms: analysis of the functional constraints imposed on replication factor C AAA+ ATPases

  2005cites this paper
• Fluorescence measurements on the E.coli DNA polymerase clamp loader: implications for conformational changes during ATP and clamp binding.

  2004influential citation
• Structural analysis of a eukaryotic sliding DNA clamp–clamp loader complex

  2004cites this paper
• The dynamic processivity of the T4 DNA polymerase during replication.

  2004cites this paper
• Secondary Structure in Protein Analysis

  2004cites this paper
• Synthesis Dependent on Genetic Recombination: acterization of a Reaction Catalyzed Purified Bacteriophage T4 Proteins

  2003cites this paper
• Protein-Protein Interactions in the Bacteriophage T4 Replisome

  2003cites this paper
• The Application of a Minicircle Substrate in the Study of the Coordinated T4 DNA Replication*

  2003cites this paper
• DNA Polymerase Sliding Clamps

  2002cites this paper
• Dissection of the ATP-driven reaction cycle of the bacteriophage T4 DNA replication processivity clamp loading system.

  2001cites this paper
• Mechanism of processivity clamp opening by the delta subunit wrench of the clamp loader complex of E. coli DNA polymerase III.

  2001cites this paper
• Crystal structure of an archaeal DNA sliding clamp: Proliferating cell nuclear antigen from Pyrococcus furiosus

  2001cites this paper
• Replisome-mediated DNA replication.

  2001cites this paper
• A Unique Organization of the Protein Subunits of the DNA Polymerase Clamp Loader in the Archaeon Methanobacterium thermoautotrophicum ΔH*

  2000cites this paper
• Characterization of bacteriophage T4-coordinated leading- and lagging-strand synthesis on a minicircle substrate.

  2000cites this paper
• The δ Subunit of DNA Polymerase III Holoenzyme Serves as a Sliding Clamp Unloader in Escherichia coli *

  2000cites this paper
• Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage.

  2000cites this paper
• Replication Slippage of Different DNA Polymerases Is Inversely Related to Their Strand Displacement Efficiency*

  1999cites this paper
• Simultaneous formation of functional leading and lagging strand holoenzyme complexes on a small, defined DNA substrate.

  1998cites this paper
• A simple mechanism for the avoidance of entanglement during chromosome replication.

  1998cites this paper
• Factors affecting inverted repeat stimulation of recombination and deletion in Saccharomyces cerevisiae.

  1998cites this paper
• DNA recognition properties of the N-terminal DNA binding domain within the large subunit of replication factor C.

  1998cites this paper
• Replication Factor C Disengages from Proliferating Cell Nuclear Antigen (PCNA) upon Sliding Clamp Formation, and PCNA Itself Tethers DNA Polymerase δ to DNA*

  1998cites this paper
• Structural Analyses of gp45 Sliding Clamp Interactions during Assembly of the Bacteriophage T4 DNA Polymerase Holoenzyme

  1997cites this paper
• Mechanism of bacteriophage T4 DNA holoenzyme assembly: the 44/62 protein acts as a molecular motor.

  1997cites this paper
• Crystal structure of the delta' subunit of the clamp-loader complex of E. coli DNA polymerase III.

  1997cites this paper
• Processivity of the Gene 41 DNA Helicase at the Bacteriophage T4 DNA Replication Fork*

  1996cites this paper
• The kinetic mechanism of formation of the bacteriophage T4 DNA polymerase sliding clamp.

  1996cites this paper
• Fluorescence monitoring of T4 polymerase holoenzyme accessory protein interactions during loading of the sliding clamp onto the template-primer junction.

  1996cites this paper
• Effect of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein on HIV-1 reverse transcriptase activity in vitro.

  1996cites this paper
• Dual role of the 44/62 protein as a matchmaker protein and DNA polymerase chaperone during assembly of the bacteriophage T4 holoenzyme complex.

  1996cites this paper
• Role of adenosine 5'-triphosphate hydrolysis in the assembly of the bacteriophage T4 DNA replication holoenzyme complex.

  1996cites this paper
• Dynamics of DNA‐tracking by two sliding‐clamp proteins.

  1996cites this paper
• Escherichia coli DNA polymerase III holoenzyme subunits alpha, beta, and gamma directly contact the primer-template.

  1995cites this paper
• Accessory proteins function as matchmakers in the assembly of the T4 DNA polymerase holoenzyme.

  1995cites this paper
• Helix-destabilizing activity of phi 29 single-stranded DNA binding protein: effect on the elongation rate during strand displacement DNA replication.

  1995cites this paper
• Escherichia coli DNA Polymerase III Holoenzyme Subunits α, β, and γ Directly Contact the Primer-Template (*)

  1995cites this paper
• Structure and Function of the UvrB Protein (*)

  1995cites this paper
• Yeast DNA repair protein RAD5 that promotes instability of simple repetitive sequences is a DNA-dependent ATPase.

  1994cites this paper
• Interaction of herpes simplex virus type 1 DNA polymerase and the UL42 accessory protein with a model primer template

  1994cites this paper
• Nucleic Acids and Molecular Biology

  1994cites this paper
• Interaction of DNA polymerase and DNA helicase within the bacteriophage T4 DNA replication complex. Leading strand synthesis by the T4 DNA polymerase mutant A737V (tsL141) requires the T4 gene 59 helicase assembly protein.

  1994cites this paper
• A single mutation in bacteriophage T4 DNA polymerase (A737V, tsL141) decreases its processivity as a polymerase and increases its processivity as a 3'-->5' exonuclease.

  1994cites this paper
• Detecting the ability of viral, bacterial and eukaryotic replication proteins to track along DNA.

  1994cites this paper
• β Sliding Clamp Dynamics within E. Coli DNA Polymerase III Holoenzyme

  1994cites this paper
• The rapid dissociation of the T4 DNA polymerase holoenzyme when stopped by a DNA hairpin helix. A model for polymerase release following the termination of each Okazaki fragment.

  1994cites this paper
• The slow dissociation of the T4 DNA polymerase holoenzyme when stalled by nucleotide omission. An indication of a highly processive enzyme.

  1994cites this paper
• The β Sliding Clamp of E. coli DNA Polymerase III Holoenzyme Balances Opposing Functions

  1994cites this paper
• Interaction ofHerpesSimplex VirusType1DNA Polymerase andthe UL42Accessory Protein witha ModelPrimer Template

  1994cites this paper
• Rapid assembly of the bacteriophage T4 core replication complex on a linear primer/template construct.

  1993cites this paper
• Molecular matchmakers.

  1993cites this paper
• Sliding clamps of DNA polymerases.

  1993cites this paper
• HeLa cell single-stranded DNA-binding protein increases the accuracy of DNA synthesis by DNA polymerase alpha in vitro.

  1992cites this paper
• Prokaryotic DNA replication.

  1992cites this paper
• Parameters that influence processive synthesis and site-specific termination by human immunodeficiency virus reverse transcriptase on RNA and DNA templates.

  1992cites this paper
• Eukaryotic DNA replication.

  1992cites this paper
• Genetic analysis of the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin.

  1992cites this paper
• Elongation factor-dependent transcript shortening by template-engaged RNA polymerase II.

  1992cites this paper
• Energy flow considerations and thermal fluctuational opening of DNA base pairs at a replicating fork: unwinding consistent with observed replication rates.

  1992cites this paper
• The sliding clamp of DNA polymerase III holoenzyme encircles DNA.

  1992cites this paper
• Structure and function of the bacteriophage T4 DNA polymerase holoenzyme.

  1992cites this paper
• Protein‐protein interactions at a DNA replication fork: bacteriophage T4 as a model

  1992cites this paper
• Cryoelectron microscopic visualization of functional subassemblies of the bacteriophage T4 DNA replication complex.

  1992cites this paper
• TheSliding ClampofDNA Polymerase IIIHoloenzyme Encircles DNA

  1992cites this paper
• Proofreading activity of DNA polymerase III responds like elongation activity to auxiliary subunits.

  1991cites this paper
• DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction.

  1991cites this paper
• Stimulation of the processivity of the DNA polymerase of bacteriophage T4 by the polymerase accessory proteins. The role of ATP hydrolysis.

  1991cites this paper
• Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis.

  1991cites this paper
• Stable RNA Secondary Structure in a Retroviral Vector Insert Terminates Reverse Transcriptase Elongation In Vitro but Not in Cultured Cells

  1990cites this paper
• Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4.

  1990cites this paper
• Functional interaction between the herpes simplex-1 DNA polymerase and UL42 protein.

  1990cites this paper
• Ordered deletions for DNA sequencing and in vitro mutagenesis by polymerase extension and exonuclease III gapping of circular templates.

  1990cites this paper