Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Exonuclease of human DNA polymerase gamma disengages its strand displacement function.

ABSTRACT: Pol ?, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol ? efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol ? has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol ? has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol ?A alone, even when exo(-), is unable to synthesize by strand displacement, making this the only known reaction of Pol ? holoenzyme that has an absolute requirement for the accessory subunit Pol ?B.

SUBMITTER: He Q

PROVIDER: S-EPMC5017585 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Exonuclease of human DNA polymerase gamma disengages its strand displacement function.

He Quan Q Shumate Christie K CK White Mark A MA Molineux Ian J IJ Yin Y Whitney YW

Mitochondrion 20130830 6

Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA ...[more]

PMID: 23993955

Similar Datasets

Strand displacement synthesis by yeast DNA polymerase ?.

Project description:DNA polymerase ? (Pol ?) is a replicative DNA polymerase with an associated 3'-5' exonuclease activity. Here, we explored the capacity of Pol ? to perform strand displacement synthesis, a process that influences many DNA transactions in vivo We found that Pol ? is unable to carry out extended strand displacement synthesis unless its 3'-5' exonuclease activity is removed. However, the wild-type Pol ? holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol ? from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ? but not with Pol ?, RB69 gp43 or Pol ?. Neither was Pol ? able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ? is distributive. Our results suggest that Pol ? is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ? participates in short-patch base excision repair and ribonucleotide excision repair.

| S-EPMC5041465 | biostudies-literature

Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1.

Project description:Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3-4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation.

| S-EPMC3783171 | biostudies-literature

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

Project description:Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.

| S-EPMC4216916 | biostudies-literature

Regulation of yeast DNA polymerase ?-mediated strand displacement synthesis by 5'-flaps.

Project description:The strand displacement activity of DNA polymerase ? is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3'-5' exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol ?-exo(-) to carry out strand displacement synthesis and discovered that it is regulated by the 5'-flaps in the DNA strand to be displaced. Under conditions where Pol ? carries out strand displacement synthesis, the presence of long 5'-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5'-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol ? with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol ? activities.

| S-EPMC4417170 | biostudies-literature

The exonuclease activity of DNA polymerase γ is required for ligation during mitochondrial DNA replication.

Project description:Mitochondrial DNA (mtDNA) polymerase γ (POLγ) harbours a 3'-5' exonuclease proofreading activity. Here we demonstrate that this activity is required for the creation of ligatable ends during mtDNA replication. Exonuclease-deficient POLγ fails to pause on reaching a downstream 5'-end. Instead, the enzyme continues to polymerize into double-stranded DNA, creating an unligatable 5'-flap. Disease-associated mutations can both increase and decrease exonuclease activity and consequently impair DNA ligation. In mice, inactivation of the exonuclease activity causes an increase in mtDNA mutations and premature ageing phenotypes. These mutator mice also contain high levels of truncated, linear fragments of mtDNA. We demonstrate that the formation of these fragments is due to impaired ligation, causing nicks near the origin of heavy-strand DNA replication. In the subsequent round of replication, the nicks lead to double-strand breaks and linear fragment formation.

| S-EPMC4557304 | biostudies-literature

A specific subdomain in phi29 DNA polymerase confers both processivity and strand-displacement capacity.

Project description:Recent crystallographic studies of phi29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a phi29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of phi29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity.

| S-EPMC1088371 | biostudies-literature

Networked Communication between Polymerase and Exonuclease Active Sites in Human Mitochondrial DNA Polymerase.

Project description:High fidelity human mitochondrial DNA polymerase (Pol ?) contains two active sites, a DNA polymerization site (pol) and a 3'-5' exonuclease site (exo) for proofreading. Although separated by 35 Å, coordination between the pol and exo sites is crucial to high fidelity replication. The biophysical mechanisms for this coordination are not completely understood. To understand the communication between the two active sites, we used a statistical-mechanical model of the protein ensemble to calculate the energetic landscape and local stability. We compared a series of structures of Pol ?, complexed with primer/template DNA, and either a nucleotide substrate or a series of nucleotide analogues, which are differentially incorporated and excised by pol and exo activity. Despite the nucleotide or its analogues being bound in the pol, Pol ? residue stability varied across the protein, particularly in the exo domain. This suggests that substrate presence in the pol can be "sensed" in the exo domain. Consistent with this hypothesis, in silico mutations made in one active site mutually perturbed the energetics of the other. To identify specific regions of the polymerase that contributed to this communication, we constructed an allosteric network connectivity map that further demonstrates specific pol-exo cooperativity. Thus, a cooperative network underlies energetic connectivity. We propose that Pol ? and other dual-function polymerases exploit an energetic coupling network that facilitates domain-domain communication to enhance discrimination between correct and incorrect nucleotides.

| S-EPMC7119269 | biostudies-literature

Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ.

Project description:Using an in vitro reconstituted system in this work we provide direct evidence that the yeast repressor/activator protein 1 (Rap1), tightly bound to its consensus site, forms a strong non-polar barrier for the strand displacement activity of DNA polymerase δ. We propose that relief of inhibition may be mediated by the activity of an accessory helicase. To this end, we show that Pif1, a 5'-3' helicase, not only stimulates the strand displacement activity of Pol δ but it also allows efficient replication through the block, by removing bound Rap1 in front of the polymerase. This stimulatory activity of Pif1 is not limited to the displacement of a single Rap1 molecule; Pif1 also allows Pol δ to carry out DNA synthesis across an array of bound Rap1 molecules that mimics a telomeric DNA-protein assembly. This activity of Pif1 represents a novel function of this helicase during DNA replication.

| S-EPMC4856994 | biostudies-literature

Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase ε holoenzyme.

Project description:In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3'→5' exonuclease activity of the hPolε holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3'→5' exonuclease activity of the hPolε holoenzyme. Together, the 3'→5' exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPolε holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPolε holoenzyme an efficient and faithful replicative DNA polymerase.

| S-EPMC7863874 | biostudies-literature

Human DNA polymerase ? accommodates RNA for strand extension.

Project description:Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase ?, telomerase, and the mitochondrial human DNA polymerase (hpol) ? have been shown to tolerate an entire RNA strand. Y-family hpol ? is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol ? is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol ?, as well as another Y-family DNA polymerase, hpol ?, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol ? redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol ? in vivo.

| S-EPMC5672030 | 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