Oxidative damage diminishes mitochondrial DNA polymerase replication fidelity.
ABSTRACT: Mitochondrial DNA (mtDNA) resides in a high ROS environment and suffers more mutations than its nuclear counterpart. Increasing evidence suggests that mtDNA mutations are not the results of direct oxidative damage, rather are caused, at least in part, by DNA replication errors. To understand how the mtDNA replicase, Pol ?, can give rise to elevated mutations, we studied the effect of oxidation of Pol ? on replication errors. Pol ? is a high fidelity polymerase with polymerase (pol) and proofreading exonuclease (exo) activities. We show that Pol ? exo domain is far more sensitive to oxidation than pol; under oxidative conditions, exonuclease activity therefore declines more rapidly than polymerase. The oxidized Pol ? becomes editing-deficient, displaying a 20-fold elevated mutations than the unoxidized enzyme. Mass spectrometry analysis reveals that Pol ? exo domain is a hotspot for oxidation. The oxidized exo residues increase the net negative charge around the active site that should reduce the affinity to mismatched primer/template DNA. Our results suggest that the oxidative stress induced high mutation frequency on mtDNA can be indirectly caused by oxidation of the mitochondrial replicase.
Project description:Replication errors are the main cause of mitochondrial DNA (mtDNA) mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POL?A) of the mtDNA polymerase. Here we genomically engineer the tamas locus, encoding fly POL?A, and introduce alleles expressing exonuclease- (exo(-)) and polymerase-deficient (pol(-)) POL?A versions. The exo(-) mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol(-) mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POL?A goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POL?A mutation disease.
Project description:Mutations in mitochondrial DNA (mtDNA) are an important cause of disease and perhaps aging in human. DNA polymerase gamma (pol ?), the unique replicase inside mitochondria, plays a key role in the fidelity of mtDNA replication through selection of the correct nucleotide and 3'-5' exonuclease proofreading. For the first time, we have isolated and characterized antimutator alleles in the yeast pol ? (Mip1). These mip1 mutations, localised in the 3'-5' exonuclease and polymerase domains, elicit a 2-15 fold decrease in the frequency of mtDNA point mutations in an msh1-1 strain which is partially deficient in mtDNA mismatch-repair. In vitro experiments show that in all mutants the balance between DNA synthesis and exonucleolysis is shifted towards excision when compared to wild-type, suggesting that in vivo more opportunity is given to the editing function for removing the replicative errors. This results in partial compensation for the mismatch-repair defects and a decrease in mtDNA point mutation rate. However, in all mutants but one the antimutator trait is lost in the wild-type MSH1 background. Accordingly, the polymerases of selected mutants show reduced oligonucleotide primed M13 ssDNA synthesis and to a lesser extent DNA binding affinity, suggesting that in mismatch-repair proficient cells efficient DNA synthesis is required to reach optimal accuracy. In contrast, the Mip1-A256T polymerase, which displays wild-type like DNA synthesis activity, increases mtDNA replication fidelity in both MSH1 and msh1-1 backgrounds. Altogether, our data show that accuracy of wild-type Mip1 is probably not optimal and can be improved by specific (often conservative) amino acid substitutions that define a pol ? area including a loop of the palm subdomain, two residues near the ExoII motif and an exonuclease helix-coil-helix module in close vicinity to the polymerase domain. These elements modulate in a subtle manner the balance between DNA polymerization and excision.
Project description: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.
Project description:Many DNA polymerases (Pol) have an intrinsic 3'-->5' exonuclease (Exo) activity which corrects polymerase errors and prevents mutations. We describe a role of the 3'-->5' Exo of Pol delta as a supplement or backup for the Rad27/Fen1 5' flap endonuclease. A yeast rad27 null allele was lethal in combination with Pol delta mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease, but it was viable with mutations in other parts of Pol delta. The rad27-p allele, which has little phenotypic effect by itself, was also lethal in combination with mutations in the Pol delta Exo I and Exo II motifs. However, rad27-p Pol delta Exo III double mutants were viable. They exhibited strong synergistic increases in CAN1 duplication mutations, intrachromosomal and interchromosomal recombination, and required the wild-type double-strand break repair genes RAD50, RAD51, and RAD52 for viability. Observed effects were similar to those of the rad27-null mutant deficient in the removal of 5' flaps in the lagging strand. These results suggest that the 3'-->5' Exo activity of Pol delta is redundant with Rad27/Fen1 for creating ligatable nicks between adjacent Okazaki fragments, possibly by reducing the amount of strand-displacement in the lagging strand.
Project description:The activity of polymerase ? is complicated, involving both correct and incorrect DNA polymerization events, exonuclease activity, and the disassociation of the polymerase:DNA complex. Pausing of pol-? might increase the chance of deletion and depletion of mitochondrial DNA. We have developed a stochastic simulation of pol-? that models its activities on the level of individual nucleotides for the replication of mtDNA. This method gives us insights into the pausing of two pol-? variants: the A467T substitution that causes PEO and Alpers syndrome, and the exonuclease deficient pol-? (exo(-)) in premature aging mouse models. To measure the pausing, we analyzed simulation results for the longest time for the polymerase to move forward one nucleotide along the DNA strand. Our model of the exo(-) polymerase had extremely long pauses, with a 30 to 300-fold increase in the time required for the longest single forward step compared to the wild-type, while the naturally occurring A467T variant showed at most a doubling in the length of the pauses compared to the wild-type. We identified the cause of these differences in the polymerase pausing time to be the number of disassociations occurring in each forward step of the polymerase.
Project description:Substitutions in the exonuclease domain of DNA polymerase ? cause ultramutated human tumors. Yeast and mouse mimics of the most common variant, P286R, produce mutator effects far exceeding the effect of Pol? exonuclease deficiency. Yeast Pol?-P301R has increased DNA polymerase activity, which could underlie its high mutagenicity. We aimed to understand the impact of this increased activity on the strand-specific role of Pol? in DNA replication and the action of extrinsic correction systems that remove Pol? errors. Using mutagenesis reporters spanning a well-defined replicon, we show that both exonuclease-deficient Pol? (Pol?-exo-) and Pol?-P301R generate mutations in a strictly strand-specific manner, yet Pol?-P301R is at least ten times more mutagenic than Pol?-exo- at each location analyzed. Thus, the cancer variant remains a dedicated leading-strand polymerase with markedly low accuracy. We further show that P301R substitution is lethal in strains lacking Pol? proofreading or mismatch repair (MMR). Heterozygosity for pol2-P301R is compatible with either defect but causes strong synergistic increases in the mutation rate, indicating that Pol?-P301R errors are corrected by Pol? proofreading and MMR. These data reveal the unexpected ease with which polymerase exchange occurs in vivo, allowing Pol? exonuclease to prevent catastrophic accumulation of Pol?-P301R-generated errors on the leading strand.
Project description:Mitochondrial DNA (mtDNA) polymerase ? (POL?) 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 POL? 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.
Project description:Mutations in the human mitochondrial polymerase (polymerase-? (Pol-?)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-? with their physiological consequences, we created "humanized" yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-?. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-? suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-? mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans.
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.
Project description:Replicative DNA polymerases (Pols) frequently possess two distinct DNA processing activities: DNA synthesis (polymerization) and proofreading (3'-5' exonuclease activity). The polymerase and exonuclease reactions are performed alternately and are spatially separated in different protein domains. Thus, the growing DNA primer terminus has to undergo dynamic conformational switching between two distinct functional sites on the polymerase. Furthermore, the transition from polymerization (pol) mode to exonuclease (exo) mode must occur in the context of a DNA Pol holoenzyme, wherein the polymerase is physically associated with processivity factor proliferating cell nuclear antigen (PCNA) and primer-template DNA. The mechanism of this conformational switching and the role that PCNA plays in it have remained obscure, largely due to the dynamic nature of ternary Pol/PCNA/DNA assemblies. Here, we present computational models of ternary assemblies for archaeal polymerase PolB. We have combined all available structural information for the binary complexes with electron microscopy data and have refined atomistic models for ternary PolB/PCNA/DNA assemblies in pol and exo modes using molecular dynamics simulations. In addition to the canonical PIP-box/interdomain connector loop (IDCL) interface of PolB with PCNA, contact analysis of the simulation trajectories revealed new secondary binding interfaces, distinct between the pol and exo states. Using targeted molecular dynamics, we explored the conformational transition from pol to exo mode. We identified a hinge region between the thumb and palm domains of PolB that is critical for conformational switching. With the thumb domain anchored onto the PCNA surface, the neighboring palm domain executed rotational motion around the hinge, bringing the core of PolB down toward PCNA to form a new interface with the clamp. A helix from PolB containing a patch of arginine residues was involved in the binding, locking the complex in the exo mode conformation. Together, these results provide a structural view of how the transition between the pol and exo states of PolB is coordinated through PCNA to achieve efficient proofreading.