Browse
Submit Data
Databases
API
Help

Dataset Information

19 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A new paradigm for DNA polymerase specificity.

ABSTRACT: We show that T7 DNA polymerase exists in three distinct structural states, as reported by a conformationally sensitive fluorophore attached to the recognition (fingers) domain. The conformational change induced by a correct nucleotide commits the substrate to the forward reaction, and the slow reversal of the conformational change eliminates the rate of the chemistry step from any contribution toward enzyme specificity. Discrimination against mismatches is enhanced by the rapid release of mismatched nucleotides from the ternary E.DNA.deoxynucleoside triphosphate complex and by the use of substrate-binding energy to actively misalign catalytic residues to reduce the rate of misincorporation. Our refined model for enzyme selectivity extends traditional thermodynamic formalism by including substrate-induced structural alignment or misalignment of catalytic residues as a third dimension on the free-energy profile and by including the rate of substrate dissociation as a key kinetic parameter.

SUBMITTER: Tsai YC

PROVIDER: S-EPMC7526746 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A new paradigm for DNA polymerase specificity.

Tsai Yu-Chih YC Johnson Kenneth A KA

Biochemistry 20060801 32

We show that T7 DNA polymerase exists in three distinct structural states, as reported by a conformationally sensitive fluorophore attached to the recognition (fingers) domain. The conformational change induced by a correct nucleotide commits the substrate to the forward reaction, and the slow reversal of the conformational change eliminates the rate of the chemistry step from any contribution toward enzyme specificity. Discrimination against mismatches is enhanced by the rapid release of mismat ...[more]

PMID: 16893169

Similar Datasets

Mammalian DNA Polymerase Kappa Activity and Specificity.

Project description:DNA polymerase (pol) kappa is a Y-family translesion DNA polymerase conserved throughout all domains of life. Pol kappa is special6 ized for the ability to copy DNA containing minor groove DNA adducts, especially N2-dG adducts, as well as to extend primer termini containing DNA damage or mismatched base pairs. Pol kappa generally cannot copy DNA containing major groove modifications or UV-induced photoproducts. Pol kappa can also copy structured or non-B-form DNA, such as microsatellite DNA, common fragile sites, and DNA containing G quadruplexes. Thus, pol kappa has roles both in maintaining and compromising genomic integrity. The expression of pol kappa is altered in several different cancer types, which can lead to genome instability. In addition, many cancer-associated single-nucleotide polymorphisms have been reported in the POLK gene, some of which are associated with poor survival and altered chemotherapy response. Because of this, identifying inhibitors of pol kappa is an active area of research. This review will address these activities of pol kappa, with a focus on lesion bypass and cellular mutagenesis.

| S-EPMC6695781 | biostudies-literature

Synthetic nucleotides as probes of DNA polymerase specificity.

Project description:The genetic code is continuously expanding with new nucleobases designed to suit specific research needs. These synthetic nucleotides are used to study DNA polymerase dynamics and specificity and may even inhibit DNA polymerase activity. The availability of an increasing chemical diversity of nucleotides allows questions of utilization by different DNA polymerases to be addressed. Much of the work in this area deals with the A family DNA polymerases, for example, Escherichia coli DNA polymerase I, which are DNA polymerases involved in replication and whose fidelity is relatively high, but more recent work includes other families of polymerases, including the Y family, whose members are known to be error prone. This paper focuses on the ability of DNA polymerases to utilize nonnatural nucleotides in DNA templates or as the incoming nucleoside triphosphates. Beyond the utility of nonnatural nucleotides as probes of DNA polymerase specificity, such entities can also provide insight into the functions of DNA polymerases when encountering DNA that is damaged by natural agents. Thus, synthetic nucleotides provide insight into how polymerases deal with nonnatural nucleotides as well as into the mutagenic potential of nonnatural nucleotides.

| S-EPMC3377560 | biostudies-literature

DNA polymerase beta substrate specificity: side chain modulation of the "A-rule".

Project description:Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of DNA polymerase (pol) beta, a family X polymerase, with active site mismatched nascent base pairs indicate that the templating (i.e. coding) base is repositioned outside of the template binding pocket thereby diminishing interactions with the incorrect incoming nucleotide. This effectively produces an abasic site because the template pocket is devoid of an instructional base. However, the template pocket is not empty; an arginine residue (Arg-283) occupies the space vacated by the templating nucleotide. In this study, we analyze the kinetics of pol beta insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant. The crystallographic structures of pol beta bound to gapped DNA with an AP site analog (tertrahydrofuran) in the gap (binary complex) and with an incoming nonhydrolyzable dATP analog (ternary complex) were solved. These structures reveal that binding of the dATP analog induces a closed polymerase conformation, an unstable primer terminus, and an upstream shift of the templating residue even in the absence of a template base. Thus, dATP insertion opposite an abasic site and dATP misinsertions have common features.

| S-EPMC2797239 | biostudies-literature

The Significance of Targeting Poly (ADP-Ribose) Polymerase-1 in Pancreatic Cancer for Providing a New Therapeutic Paradigm.

Project description:Genome-wide studies focusing on elucidating the effects on cancer progression have enabled the consequent identification of a distinct subpopulation of pancreatic cancer cells with unstable genomic characteristics. Based on this background, deleterious changes by poly (adenosine diphosphate (ADP)-ribose) polymerase-1 (PARP)-1 have been concentrated in oncology. One of the critical functions of PARP-1 is the response to DNA damage, which plays a pivotal role in DNA repair in cancers. PARP-1 also has widespread functions that are essential for the survival and growth of cancer cells. It regulates oxidative stress in mitochondria through the regulation of superoxide and oxidation. PARP-1 is in charge of regulating mitosis, which is a crucial role in tumorigenesis and remodels histones and chromatin enzymes related to transcriptional regulation, causing alterations in epigenetic markers and chromatin structure. Given the significance of these processes, it can be understood that these processes in cancer cells are at the frontline of the pathogenetic changes required for cancer cell survival, and these contributions can result in malignant transformation. Therefore, this review addresses the current molecular biological features for understanding the multifactorial function of PARP-1 in pancreatic cancer related to the aforementioned roles, along with the summary of recent approaches with PARP-1 inhibition in clinical studies targeting pancreatic cancer. This understanding could help to embrace the importance of targeting PARP-1 in the treatment of pancreatic cancer, which may present the potential to find out a variety of research topics that can be both challenged clinically and non-clinically.

| S-EPMC8037971 | biostudies-literature

A new proofreading mechanism for lesion bypass by DNA polymerase-?.

Project description:Replicative DNA polymerases (DNA pols) increase their fidelity by removing misincorporated nucleotides with their 3' ? 5' exonuclease activity. Exonuclease activity reduces translesion synthesis (TLS) efficiency and TLS DNA pols lack 3' ? 5' exonuclease activity. Here we show that physiological concentrations of pyrophosphate (PP(i)) activate the pyrophosphorolytic activity by DNA pol-?, allowing the preferential excision of the incorrectly incorporated A opposite a 7,8-dihydro-8-oxoguanine lesion, or T opposite a 6-methyl-guanine, with respect to the correct C. This is the first example of an alternative proofreading mechanism used during TLS.

| S-EPMC3246252 | biostudies-literature

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8.

Project description:All genetic information in cellular life is stored in DNA copolymers composed of four basic building blocks (ATGC-DNA). In contrast, a group of bacteriophages belonging to families Siphoviridae and Podoviridae has abandoned the usage of one of them, adenine (A), replacing it with 2-aminoadenine (Z). The resulting ZTGC-DNA is more stable than its ATGC-DNA counterpart, owing to the additional hydrogen bond present in the 2-aminoadenine:thymine (Z:T) base pair, while the additional amino group also confers resistance to the host endonucleases. Recently, two classes of replicative proteins found in ZTGC-DNA-containing phages were characterized and one of them, DpoZ from DNA polymerase A (PolA) family, was shown to possess significant Z-vs-A specificity. Here, we present the crystallographic structure of the apo form of DpoZ of vibriophage ϕVC8, composed of the 3'-5' exonuclease and polymerase domains. We captured the enzyme in two conformations that involve the tip of the thumb subdomain and the exonuclease domain. We highlight insertions and mutations characteristic of ϕVC8 DpoZ and its close homologues. Through mutagenesis and functional assays we suggest that the preference of ϕVC8 DpoZ towards Z relies on a polymerase backtracking process, more efficient when the nascent base pair is A:T than when it is Z:T.

| S-EPMC8599892 | biostudies-literature

Effects of Active Site Mutations on Specificity of Nucleobase Binding in Human DNA Polymerase η.

Project description:Human DNA polymerase η (Pol η) plays a vital role in protection against skin cancer caused by damage from ultraviolet light. This enzyme rescues stalled replication forks at cyclobutane thymine-thymine dimers (TTDs) by inserting nucleotides opposite these DNA lesions. Residue R61 is conserved in the Pol η enzymes across species, but the corresponding residue, as well as its neighbor S62, is different in other Y-family polymerases, Pol ι and Pol κ. Herein, R61 and S62 are mutated to their Pol ι and Pol κ counterparts. Relative binding free energies of dATP to mutant Pol η•DNA complexes with and without a TTD were calculated using thermodynamic integration. The binding free energies of dATP to the Pol η•DNA complex with and without a TTD are more similar for all of these mutants than for wild-type Pol η, suggesting that these mutations decrease the ability of this enzyme to distinguish between a TTD lesion and undamaged DNA. Molecular dynamics simulations of the mutant systems provide insights into the molecular level basis for the changes in relative binding free energies. The simulations identified differences in hydrogen-bonding, cation-π, and π-π interactions of the side chains with the dATP and the TTD or thymine-thymine (TT) motif. The simulations also revealed that R61 and Q38 act as a clamp to position the dATP and the TTD or TT and that the mutations impact the balance among the interactions related to this clamp. Overall, these calculations suggest that R61 and S62 play key roles in the specificity and effectiveness of Pol η for bypassing TTD lesions during DNA replication. Understanding the basis for this specificity is important for designing drugs aimed at cancer treatment.

| S-EPMC5402696 | biostudies-literature

DNA polymerase beta: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes.

Project description:Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.

| S-EPMC1219323 | biostudies-other

Involvement of phage phi29 DNA polymerase and terminal protein subdomains in conferring specificity during initiation of protein-primed DNA replication.

Project description:To initiate phi29 DNA replication, the DNA polymerase has to form a complex with the homologous primer terminal protein (TP) that further recognizes the replication origins of the homologous TP-DNA placed at both ends of the linear genome. By means of chimerical proteins, constructed by swapping the priming domain of the related phi29 and GA-1 TPs, we show that DNA polymerase can form catalytically active heterodimers exclusively with that chimerical TP containing the N-terminal part of the homologous TP, suggesting that the interaction between the polymerase TPR-1 subdomain and the TP N-terminal part is the one mainly responsible for the specificity between both proteins. We also show that the TP N-terminal part assists the proper binding of the priming domain at the polymerase active site. Additionally, a chimerical 29 DNA polymerase containing the GA-1 TPR-1 subdomain could use GA-1 TP, but only in the presence of phi29 TP-DNA as template, indicating that parental TP recognition is mainly accomplished by the DNA polymerase. The sequential events occurring during initiation of bacteriophage protein-primed DNA replication are proposed.

| S-EPMC2175359 | biostudies-literature

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis.

Project description:Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ ions in each step in the catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. An increase of the Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases.

| S-EPMC7948414 | 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