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

Macao B, Uhler JP, Siibak T, Zhu X, Shi Y, Sheng W, Olsson M, Stewart JB, Gustafsson CM, Falkenberg M - Nat Commun (2015)

Bottom Line: Disease-associated mutations can both increase and decrease exonuclease activity and consequently impair DNA ligation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Medicinaregatan 9 A, P.O. Box 440, SE-40530 Gothenburg, Sweden.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

DNA exonuclease and polymerization activities of POLγ mutants.(a) Schematic diagram of the exonuclease domain of POLγA and the position of the patient-associated mutations. The EXO- mutation D274A is indicated in grey. Black boxes represent the exonuclease motifs I, II and III. (b) The DNA-binding efficiencies of the POLγA mutants were tested on a primed template either as monomers or heterotrimers with POLγB (as illustrated on the left side). The R275Q, G303R, L304R and S305R mutants had markedly reduced binding activity as monomers. (c) Similar to EXO- POLγ, the G303R, L304R and S305R mutants have no apparent exonuclease activity. R232H has increased exonuclease activity compared with WT. The substrate is shown in the lower right quadrant. (d) DNA polymerization by the different POLγ proteins on a circular, 3,000-nt ssDNA template (as shown in bottom right quadrant). Synthesis rates are moderately slower for the R232H and R275Q mutants, and markedly compromised for the G303R, L304R and S305R proteins.
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f3: DNA exonuclease and polymerization activities of POLγ mutants.(a) Schematic diagram of the exonuclease domain of POLγA and the position of the patient-associated mutations. The EXO- mutation D274A is indicated in grey. Black boxes represent the exonuclease motifs I, II and III. (b) The DNA-binding efficiencies of the POLγA mutants were tested on a primed template either as monomers or heterotrimers with POLγB (as illustrated on the left side). The R275Q, G303R, L304R and S305R mutants had markedly reduced binding activity as monomers. (c) Similar to EXO- POLγ, the G303R, L304R and S305R mutants have no apparent exonuclease activity. R232H has increased exonuclease activity compared with WT. The substrate is shown in the lower right quadrant. (d) DNA polymerization by the different POLγ proteins on a circular, 3,000-nt ssDNA template (as shown in bottom right quadrant). Synthesis rates are moderately slower for the R232H and R275Q mutants, and markedly compromised for the G303R, L304R and S305R proteins.

Mentions: A number of disease-causing amino acid substitutions have been identified in the exonuclease domain of POLγ31 and we wanted to investigate whether these mutations also affected exonuclease activity and the formation of ligatable ends. To this end, we expressed and purified seven mutant POLγA variants in recombinant form (R232H, G268A, R275Q, H277L, G303R, L304R and S305R (Fig. 3a)). We first investigated the DNA-binding activity of the mutants in an electrophoretic mobility shift assay (EMSA), in which POLγA was incubated together with a short, radioactively labelled primed DNA template. In the absence of POLγB, the R275Q, G303R, L304R and S305R mutants all displayed reduced DNA-binding affinity compared with WT POLγ (Fig. 3b). In combination with POLγB however, all the mutants were able to bind DNA (Fig. 3b) and to synthesize a short stretch of DNA with efficiency similar to that observed for WT POLγ (Supplementary Fig. 4).


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

Macao B, Uhler JP, Siibak T, Zhu X, Shi Y, Sheng W, Olsson M, Stewart JB, Gustafsson CM, Falkenberg M - Nat Commun (2015)

DNA exonuclease and polymerization activities of POLγ mutants.(a) Schematic diagram of the exonuclease domain of POLγA and the position of the patient-associated mutations. The EXO- mutation D274A is indicated in grey. Black boxes represent the exonuclease motifs I, II and III. (b) The DNA-binding efficiencies of the POLγA mutants were tested on a primed template either as monomers or heterotrimers with POLγB (as illustrated on the left side). The R275Q, G303R, L304R and S305R mutants had markedly reduced binding activity as monomers. (c) Similar to EXO- POLγ, the G303R, L304R and S305R mutants have no apparent exonuclease activity. R232H has increased exonuclease activity compared with WT. The substrate is shown in the lower right quadrant. (d) DNA polymerization by the different POLγ proteins on a circular, 3,000-nt ssDNA template (as shown in bottom right quadrant). Synthesis rates are moderately slower for the R232H and R275Q mutants, and markedly compromised for the G303R, L304R and S305R proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557304&req=5

f3: DNA exonuclease and polymerization activities of POLγ mutants.(a) Schematic diagram of the exonuclease domain of POLγA and the position of the patient-associated mutations. The EXO- mutation D274A is indicated in grey. Black boxes represent the exonuclease motifs I, II and III. (b) The DNA-binding efficiencies of the POLγA mutants were tested on a primed template either as monomers or heterotrimers with POLγB (as illustrated on the left side). The R275Q, G303R, L304R and S305R mutants had markedly reduced binding activity as monomers. (c) Similar to EXO- POLγ, the G303R, L304R and S305R mutants have no apparent exonuclease activity. R232H has increased exonuclease activity compared with WT. The substrate is shown in the lower right quadrant. (d) DNA polymerization by the different POLγ proteins on a circular, 3,000-nt ssDNA template (as shown in bottom right quadrant). Synthesis rates are moderately slower for the R232H and R275Q mutants, and markedly compromised for the G303R, L304R and S305R proteins.
Mentions: A number of disease-causing amino acid substitutions have been identified in the exonuclease domain of POLγ31 and we wanted to investigate whether these mutations also affected exonuclease activity and the formation of ligatable ends. To this end, we expressed and purified seven mutant POLγA variants in recombinant form (R232H, G268A, R275Q, H277L, G303R, L304R and S305R (Fig. 3a)). We first investigated the DNA-binding activity of the mutants in an electrophoretic mobility shift assay (EMSA), in which POLγA was incubated together with a short, radioactively labelled primed DNA template. In the absence of POLγB, the R275Q, G303R, L304R and S305R mutants all displayed reduced DNA-binding affinity compared with WT POLγ (Fig. 3b). In combination with POLγB however, all the mutants were able to bind DNA (Fig. 3b) and to synthesize a short stretch of DNA with efficiency similar to that observed for WT POLγ (Supplementary Fig. 4).

Bottom Line: Disease-associated mutations can both increase and decrease exonuclease activity and consequently impair DNA ligation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Medicinaregatan 9 A, P.O. Box 440, SE-40530 Gothenburg, Sweden.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus