Limits...
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 strand-displacement and ligation activity of POLγ mutants.(a) Time course of strand displacement by purified recombinant WT and mutant POLγ proteins. The reactions were performed as depicted in Fig. 1a. Arrows: solid lines indicate gap filling; broken lines indicate strand displacement. Lanes are numbered 1–54. (b) Strand-displacement reactions as above resolved on a sequencing gel. The failure by G303R, L304R and S305R to reverse to the nick position at the 10 min time point is evident (arrowhead at 50 nts). The sizes of an oligonucleotide molecular marker are indicated to the left. (c) Quantification of ligation efficiencies (ligation product formed as percentage of substrate) of the different POLγ proteins relative to WT. The ligation assay and quantification was performed as in Fig. 2c–e. Mean values±s.e.m., WT was set to 100%, asterisks represent significant differences compared with WT (n=3; *P≤0.05, **P≤0.01, ***P≤0.001; one way analysis of variance).
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f4: DNA strand-displacement and ligation activity of POLγ mutants.(a) Time course of strand displacement by purified recombinant WT and mutant POLγ proteins. The reactions were performed as depicted in Fig. 1a. Arrows: solid lines indicate gap filling; broken lines indicate strand displacement. Lanes are numbered 1–54. (b) Strand-displacement reactions as above resolved on a sequencing gel. The failure by G303R, L304R and S305R to reverse to the nick position at the 10 min time point is evident (arrowhead at 50 nts). The sizes of an oligonucleotide molecular marker are indicated to the left. (c) Quantification of ligation efficiencies (ligation product formed as percentage of substrate) of the different POLγ proteins relative to WT. The ligation assay and quantification was performed as in Fig. 2c–e. Mean values±s.e.m., WT was set to 100%, asterisks represent significant differences compared with WT (n=3; *P≤0.05, **P≤0.01, ***P≤0.001; one way analysis of variance).

Mentions: Whereas the strand-displacement activity of EXO- polymerase was clearly visible in the long-stretch DNA synthesis assay (Fig. 3d, smear above main product in lanes 8–10), we could not see a similar effect for the disease-causing mutants. We decided to further analyse the strand-displacement activities of the different mutant polymerases over time, using the linear gapped substrate (shown in Fig. 1a). Precise gap filling will result in a band of 50 nt, whereas strand-displacement will generate products up to 80 nt. Using this template, none of the patient-related mutant proteins had strand-displacement activities as severe as the EXO- (Fig. 4a). We did however notice that three mutants, L304R, G303R and S305R, generated bands around 50 nts that appeared broader than the corresponding band generated by the WT (Fig. 4a, compare lanes 4–6 with 34–36, 46–48 and 52–54), which could be the result of entry into the duplex region and the creation of a short 5′-flap. To address this possibility, we used sequencing gels for increased resolution (Fig. 4b). In this analysis, we observed stalling at position 50–56 (that is, at the nick or 1–6 nt within the downstream dsDNA region). In keeping with its increased exonuclease activity, the R232H mutant displayed reduced strand-displacement activity, pausing at the nick (Fig. 4b, compare lanes 2–3 with 6–7). The H277L, G268A and R275Q mutants behaved similar to WT POLγ by pausing at the nick position or 1–2 nt downstream. The remaining three mutants, L304R, G303R and S305R (lanes 12-13, 16–19), stalled in a broader range within the downstream dsDNA region, creating longer 5′-flaps.


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 strand-displacement and ligation activity of POLγ mutants.(a) Time course of strand displacement by purified recombinant WT and mutant POLγ proteins. The reactions were performed as depicted in Fig. 1a. Arrows: solid lines indicate gap filling; broken lines indicate strand displacement. Lanes are numbered 1–54. (b) Strand-displacement reactions as above resolved on a sequencing gel. The failure by G303R, L304R and S305R to reverse to the nick position at the 10 min time point is evident (arrowhead at 50 nts). The sizes of an oligonucleotide molecular marker are indicated to the left. (c) Quantification of ligation efficiencies (ligation product formed as percentage of substrate) of the different POLγ proteins relative to WT. The ligation assay and quantification was performed as in Fig. 2c–e. Mean values±s.e.m., WT was set to 100%, asterisks represent significant differences compared with WT (n=3; *P≤0.05, **P≤0.01, ***P≤0.001; one way analysis of variance).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f4: DNA strand-displacement and ligation activity of POLγ mutants.(a) Time course of strand displacement by purified recombinant WT and mutant POLγ proteins. The reactions were performed as depicted in Fig. 1a. Arrows: solid lines indicate gap filling; broken lines indicate strand displacement. Lanes are numbered 1–54. (b) Strand-displacement reactions as above resolved on a sequencing gel. The failure by G303R, L304R and S305R to reverse to the nick position at the 10 min time point is evident (arrowhead at 50 nts). The sizes of an oligonucleotide molecular marker are indicated to the left. (c) Quantification of ligation efficiencies (ligation product formed as percentage of substrate) of the different POLγ proteins relative to WT. The ligation assay and quantification was performed as in Fig. 2c–e. Mean values±s.e.m., WT was set to 100%, asterisks represent significant differences compared with WT (n=3; *P≤0.05, **P≤0.01, ***P≤0.001; one way analysis of variance).
Mentions: Whereas the strand-displacement activity of EXO- polymerase was clearly visible in the long-stretch DNA synthesis assay (Fig. 3d, smear above main product in lanes 8–10), we could not see a similar effect for the disease-causing mutants. We decided to further analyse the strand-displacement activities of the different mutant polymerases over time, using the linear gapped substrate (shown in Fig. 1a). Precise gap filling will result in a band of 50 nt, whereas strand-displacement will generate products up to 80 nt. Using this template, none of the patient-related mutant proteins had strand-displacement activities as severe as the EXO- (Fig. 4a). We did however notice that three mutants, L304R, G303R and S305R, generated bands around 50 nts that appeared broader than the corresponding band generated by the WT (Fig. 4a, compare lanes 4–6 with 34–36, 46–48 and 52–54), which could be the result of entry into the duplex region and the creation of a short 5′-flap. To address this possibility, we used sequencing gels for increased resolution (Fig. 4b). In this analysis, we observed stalling at position 50–56 (that is, at the nick or 1–6 nt within the downstream dsDNA region). In keeping with its increased exonuclease activity, the R232H mutant displayed reduced strand-displacement activity, pausing at the nick (Fig. 4b, compare lanes 2–3 with 6–7). The H277L, G268A and R275Q mutants behaved similar to WT POLγ by pausing at the nick position or 1–2 nt downstream. The remaining three mutants, L304R, G303R and S305R (lanes 12-13, 16–19), stalled in a broader range within the downstream dsDNA region, creating longer 5′-flaps.

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