Browse
Submit Data
Databases
API
Help

Dataset Information

24 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The kinetic and chemical mechanism of high-fidelity DNA polymerases.

ABSTRACT: This review summarizes our current understanding of the structural, kinetic and thermodynamic basis for the extraordinary accuracy of high-fidelity DNA polymerases. High-fidelity DNA polymerases, such as the enzyme responsible for the replication of bacteriophage T7 DNA, discriminate against similar substrates with an accuracy that approaches one error in a million base pairs while copying DNA at a rate of approximately 300 base pairs per second. When the polymerase does make an error, it stalls, giving time for the slower proofreading exonuclease to remove the mismatch so that the overall error frequency approaches one in a billion. Structural analysis reveals a large change in conformation after nucleotide binding from an open to a closed state. Kinetic analysis has shown that the substrate-induced structural change plays a key role in the discrimination between correct and incorrect base pairs by governing whether a nucleotide will be retained and incorporated or rapidly released.

SUBMITTER: Johnson KA

PROVIDER: S-EPMC3047511 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

The kinetic and chemical mechanism of high-fidelity DNA polymerases.

Johnson Kenneth A KA

Biochimica et biophysica acta 20100115 5

This review summarizes our current understanding of the structural, kinetic and thermodynamic basis for the extraordinary accuracy of high-fidelity DNA polymerases. High-fidelity DNA polymerases, such as the enzyme responsible for the replication of bacteriophage T7 DNA, discriminate against similar substrates with an accuracy that approaches one error in a million base pairs while copying DNA at a rate of approximately 300 base pairs per second. When the polymerase does make an error, it stalls ...[more]

PMID: 20079883

Similar Datasets

Lesion processing: high-fidelity versus lesion-bypass DNA polymerases.

Project description:When a high-fidelity DNA polymerase encounters certain DNA-damage sites, its progress can be stalled and one or more lesion-bypass polymerases are recruited to transit the lesion. Here, we consider two representative types of lesions: (i) 7,8-dihydro-8-oxoguanine (8-oxoG), a small, highly prevalent lesion caused by oxidative damage; and (ii) bulky lesions derived from the environmental pre-carcinogen benzo[a]pyrene, in the high-fidelity DNA polymerase Bacillus fragment (BF) from Bacillus stearothermophilus and in the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. The tight fit of the BF polymerase around the nascent base pair contrasts with the more spacious, solvent-exposed active site of Dpo4, and these differences in architecture result in distinctions in their respective functions: one-step versus stepwise polymerase translocation, mutagenic versus accurate bypass of 8-oxoG, and polymerase stalling versus mutagenic bypass at bulky benzo[a]pyrene-derived lesions.

| S-EPMC2717799 | biostudies-literature

Both high-fidelity replicative and low-fidelity Y-family polymerases are involved in DNA rereplication.

Project description:DNA rereplication is a major form of aberrant replication that causes genomic instabilities, such as gene amplification. However, little is known about which DNA polymerases are involved in the process. Here, we report that low-fidelity Y-family polymerases (Y-Pols), Pol η, Pol ι, Pol κ, and REV1, significantly contribute to DNA synthesis during rereplication, while the replicative polymerases, Pol δ and Pol ε, play an important role in rereplication, as expected. When rereplication was induced by depletion of geminin, these polymerases were recruited to rereplication sites in human cell lines. This finding was supported by RNA interference (RNAi)-mediated knockdown of the polymerases, which suppressed rereplication induced by geminin depletion. Interestingly, epistatic analysis indicated that Y-Pols collaborate in a common pathway, independently of replicative polymerases. We also provide evidence for a catalytic role for Pol η and the involvement of Pol η and Pol κ in cyclin E-induced rereplication. Collectively, our findings indicate that, unlike normal S-phase replication, rereplication induced by geminin depletion and oncogene activation requires significant contributions of both Y-Pols and replicative polymerases. These findings offer important mechanistic insights into cancer genomic instability.

| S-EPMC4301717 | biostudies-literature

WRN exonuclease imparts high fidelity on translesion synthesis by Y family DNA polymerases.

Project description:Purified translesion synthesis (TLS) DNA polymerases (Pols) replicate through DNA lesions with a low fidelity; however, TLS operates in a predominantly error-free manner in normal human cells. To explain this incongruity, here we determine whether Y family Pols, which play an eminent role in replication through a diversity of DNA lesions, are incorporated into a multiprotein ensemble and whether the intrinsically high error rate of the TLS Pol is ameliorated by the components in the ensemble. To this end, we provide evidence for an indispensable role of Werner syndrome protein (WRN) and WRN-interacting protein 1 (WRNIP1) in Rev1-dependent TLS by Y family Polη, Polι, or Polκ and show that WRN, WRNIP1, and Rev1 assemble together with Y family Pols in response to DNA damage. Importantly, we identify a crucial role of WRN's 3' → 5' exonuclease activity in imparting high fidelity on TLS by Y family Pols in human cells, as the Y family Pols that accomplish TLS in an error-free manner manifest high mutagenicity in the absence of WRN's exonuclease function. Thus, by enforcing high fidelity on TLS Pols, TLS mechanisms have been adapted to safeguard against genome instability and tumorigenesis.

| S-EPMC11065173 | biostudies-literature

Efficiency and fidelity of human DNA polymerases ? and ? during gap-filling DNA synthesis.

Project description:The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase ? (Pol ?), an enzyme proposed to support the known BER DNA polymerase ? (Pol ?), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol ? incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol ?, Pol ? did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol ?, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol ? was up to 160-fold more error-prone than Pol ?, thereby suggesting Pol ? would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol ? and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol ? and Pol ? would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol ?, like Pol ?, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol ? may play a role in long-patch BER.

| S-EPMC3065367 | biostudies-literature

Fidelity of DNA polymerases in the detection of intraindividual variation of mitochondrial DNA.

Project description:Here we investigated the consequences of PCR amplification errors in the identification of intraindividual mtDNA variation. The bumblebee Bombus morio was chosen as model for the COI gene amplification tests with two DNA polymerases (Taq and Q5) presenting different error rates. The amplifications using Taq resulted in a significant increase of singleton haplotypes per individual in comparison to Q5. The sequence characteristics indicated that Taq resulted haplotypes are mostly due to amplification errors. Studies focusing on intraindividual variability should address special attention to the DNA polymerase fidelity to avoid overestimation of heteroplasmic haplotypes.

| S-EPMC7720943 | biostudies-literature

Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases.

Project description:Divalent metal ions are essential components of DNA polymerases both for catalysis of the nucleotidyl transfer reaction and for base excision. They occupy two sites, A and B, for DNA synthesis. Recently, a third metal ion was shown to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by the carboxylate of two highly conserved acidic residues, water molecules, and the 3'-hydroxyl group of the primer so that the A metal is in an octahedral complex. Its catalytic function is to lower the pKa of the hydroxyl group, making it a highly effective nucleophile that can attack the α phosphorous atom of the incoming dNTP. The metal ion in the B site is coordinated by the same two carboxylates that are affixed to the A metal ion as well as the non-bridging oxygen atoms of the incoming dNTP. The carboxyl oxygen of an adjacent peptide bond serves as the sixth ligand that completes the octahedral coordination geometry of the B metal ion. Similarly, two metal ions are required for proofreading; one helps to lower the pKa of the attacking water molecule, and the other helps to stabilize the transition state for nucleotide excision. The role of different divalent cations are discussed in relation to these two activities as well as their influence on base selectivity and misincorporation by DNA polymerases. Some, but not all, of the effects of these different metal ions can be rationalized based on their intrinsic properties, which are tabulated in this review.

| S-EPMC5076500 | biostudies-literature

Structural and Molecular Kinetic Features of Activities of DNA Polymerases.

Project description:DNA polymerases catalyze DNA synthesis during the replication, repair, and recombination of DNA. Based on phylogenetic analysis and primary protein sequences, DNA polymerases have been categorized into seven families: A, B, C, D, X, Y, and RT. This review presents generalized data on the catalytic mechanism of action of DNA polymerases. The structural features of different DNA polymerase families are described in detail. The discussion highlights the kinetics and conformational dynamics of DNA polymerases from all known polymerase families during DNA synthesis.

| S-EPMC9224347 | biostudies-literature

High fidelity DNA strand-separation is the major specificity determinant in DNA methyltransferase CcrM's catalytic mechanism.

Project description:Strand-separation is emerging as a novel DNA recognition mechanism but the underlying mechanisms and quantitative contribution of strand-separation to fidelity remain obscure. The bacterial DNA adenine methyltransferase, CcrM, recognizes 5'GANTC'3 sequences through a DNA strand-separation mechanism with unusually high selectivity. To explore this novel recognition mechanism, we incorporated Pyrrolo-dC into cognate and noncognate DNA to monitor the kinetics of strand-separation and used tryptophan fluorescence to follow protein conformational changes. Both signals are biphasic and global fitting showed that the faster phase of DNA strand-separation was coincident with the protein conformational transition. Non-cognate sequences did not display strand-separation and methylation was reduced > 300-fold, providing evidence that strand-separation is a major determinant of selectivity. Analysis of an R350A mutant showed that the enzyme conformational step can occur without strand-separation, so the two events are uncoupled. A stabilizing role for the methyl-donor (SAM) is proposed; the cofactor interacts with a critical loop which is inserted between the DNA strands, thereby stabilizing the strand-separated conformation. The results presented here are broadly applicable to the study of other N6-adenine methyltransferases that contain the structural features implicated in strand-separation, which are found widely dispersed across many bacterial phyla, including human and animal pathogens, and some Eukaryotes.

| S-EPMC10359602 | biostudies-literature

Mechanism of strand displacement synthesis by DNA replicative polymerases.

Project description:Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing.

| S-EPMC3401438 | biostudies-literature

Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism.

Project description:The number of DNA polymerases identified in each organism has mushroomed in the past two decades. Most newly found DNA polymerases specialize in translesion synthesis and DNA repair instead of replication. Although intrinsic error rates are higher for translesion and repair polymerases than for replicative polymerases, the specialized polymerases increase genome stability and reduce tumorigenesis. Reflecting the numerous types of DNA lesions and variations of broken DNA ends, translesion and repair polymerases differ in structure, mechanism, and function. Here, we review the unique and general features of polymerases specialized in lesion bypass, as well as in gap-filling and end-joining synthesis.

| S-EPMC6098713 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data