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Biochim Biophys Acta
2011 Dec 01;181312:2000-7. doi: 10.1016/j.bbamcr.2011.08.008.
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Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells.
Ruhanen H
,
Ushakov K
,
Yasukawa T
.
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Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.
Fig. 1. Knockdown of DNA ligase III delays the recovery of mtDNA copy number. (A) Experimental design. The presence and absence of 2â²,3â²-dideoxycytidine (ddC) in the medium is indicated by dark and pale grey stripes respectively. (a) Time course of the ddC treatment and dsRNA knockdown. R2 and R3 represent the cell harvest points 2 and 3 days after ddC removal, whilst an arrowhead indicates the timing of dsRNA transfection. (b and c) Incubation of cells in medium in the absence or presence of ddC for 3 days as the non-treated or ddC-treated controls. NT and D3 represent the point of their harvest. (B) Western blot analysis of DNA ligase III levels in cell lysates prepared from cells treated with scramble (Sc) dsRNA or DNA ligase III-specific (LIII) dsRNA. The top panel shows the DNA ligase III band and the middle panel is a digitally enhanced image of the top panel. Tubulin was used as a loading control (bottom panel). (C) The relative copy number of mtDNA analysed with real-time quantitative PCR. The mtDNA content was normalised against nuclear gene content in each sample. The relative mtDNA copy number in NT sample was expressed as 100 in each experiment and those of the other samples were displayed relative to this. Data represent the mean of 3 independent transfection experiments ± SEM. * (p < 0.05).
Fig. 2. Sensitivity of mtDNA replication intermediates to S1 nuclease upon DNA ligase III depletion. (A) Analysis of a DraI-digested fragment of mtDNA with two-dimensional agarose gel electrophoresis. (a) A schematic drawing of the fragment visualised by Southern hybridisation. The non-replicating molecules (1N spot) and replication intermediates (Y arc) are indicated. The molecule structure of the DraI-digested mtDNA replication intermediates at 3 different positions of Y arc is drawn in the insets with a black bar as non-replicating portion and grey bars as replicated portions of the fragment. The apex region of the Y arc used for the quantification is indicated as a grey rectangle and the region of 1N spot used for the quantification as a grey dotted circle. More information on the fragment is provided in Supplementary Fig. 1B. (bâe) The DraI-digested mtDNA fragment of scramble (Sc) dsRNA or DNA ligase III-specific (LIII) dsRNA-treated cells without (â S1) (b and c) and with S1 nuclease treatment (+ S1) (d and e). The samples in panels bâe were run in the same second dimension gel and panels bâe were produced from an X-ray film. Panels câ² and eâ² are a longer exposure version of panels c and e. (B) Numerical presentation of the Y arc stability against S1 nuclease. The value of (y/1n [+ S1])/(y/1n [â S1]) from Sc dsRNA-treated samples is expressed as 100 and the relative value from LIII dsRNA-treated samples was calculated. Data represent the mean of 3 independent transfection experiments ± SEM.Sensitivity of mtDNA replication intermediates to S1 nuclease upon DNA ligase III depletion. (A) Analysis of a DraI-digested fragment of mtDNA with two-dimensional agarose gel electrophoresis. (a) A schematic drawing of the fragment visualised by Southern hybridisation. The non-replicating molecules (1N spot) and replication intermediates (Y arc) are indicated. The molecule structure of the DraI-digested mtDNA replication intermediates at 3 different positions of Y arc is drawn in the insets with a black bar as non-replicating portion and grey bars as replicated portions of the fragment. The apex region of the Y arc used for the quantification is indicated as a grey rectangle and the region of 1N spot used for the quantification as a grey dotted circle. More information on the fragment is provided in Supplementary Fig. 1B. (bâe) The DraI-digested mtDNA fragment of scramble (Sc) dsRNA or DNA ligase III-specific (LIII) dsRNA-treated cells without (â S1) (b and c) and with S1 nuclease treatment (+ S1) (d and e). The samples in panels bâe were run in the same second dimension gel and panels bâe were produced from an X-ray film. Panels câ² and eâ² are a longer exposure version of panels c and e. (B) Numerical presentation of the Y arc stability against S1 nuclease. The value of (y/1n [+ S1])/(y/1n [â S1]) from Sc dsRNA-treated samples is expressed as 100 and the relative value from LIII dsRNA-treated samples was calculated. Data represent the mean of 3 independent transfection experiments ± SEM.
Fig. 3. Knockdown of RNase H1, but neither FEN1 nor DNA2 inhibits mtDNA replication. Cells were transiently treated with 2â²,3â²-dideoxycytidine and transfected with scramble (Sc) dsRNA, RNase H1-specific (RH1) dsRNA, FEN1-specific (FEN1) dsRNA, DNA2-specific (DNA2) dsRNA and both FEN1 dsRNA and DNA2 dsRNA (F + D) as in the diagram of Fig. 1A. (A) Western blot analysis of RNase H1, FEN1 and DNA2 levels. Below each panel Tubulin is shown as loading control. (B) The relative copy number of mtDNA analysed with real-time quantitative qPCR. The interpretation of the graph is as in Fig. 1C. Data represent the mean of 3 independent transfection experiments ± S.E.M. * (p < 0.05).
Fig. 4. RNase H1 depletion causes enhanced sensitivity to S1 nuclease of mtDNA replication intermediates. (A) Analysis of a DraI-digested mtDNA fragment as in Fig. 2A from cells treated with scramble (Sc) dsRNA or RNase H1-specific (RH1) dsRNA (â S1) (a and b) and those with S1 nuclease treatment (+ S1) (c and d). The images were produced from a single membrane exposed to an X-ray film. (B) Numerical presentation of the Y arc stability against S1 nuclease upon knockdown with RH1 dsRNA. The presentation of the graph is the same as in Fig. 2B. Data represent the mean of 3 independent transfection experiments ± S.E.M. One of the three transfection experiments in Fig. 3B was not used for the production of this graph. Thus the 2D-AGE image from an additional transfection experiment was used instead. ** (p < 0.01).
Fig. 5. Knockdown of FEN1 and DNA2 does not increase the sensitivity of mtDNA replication intermediates to S1 nuclease. Two-dimensional agarose gel electrophoresis analysis of a DraI-digested mtDNA fragment. Samples were prepared from cells transfected with scramble (Sc) dsRNA, FEN1-specific (FEN1) dsRNA, DNA2-specific (DNA2) dsRNA or both (F + D). (A) Panels a, b and c are samples transfected with Sc, FEN1 and DNA2 dsRNA without S1 nuclease treatment (â S1) respectively, and panels d, e, and f are those with S1 nuclease treatment (+ S1) respectively. Panels aâf were produced from an X-ray film. (B) Panels g and h are samples with the control and double knockdown of FEN1 and DNA2 without S1 nuclease treatment (â S1) respectively, and panels i and j are those with S1 nuclease treatment (+ S1) respectively. Panels gâj were produced from an X-ray film. (C and D) Numerical presentation of the Y arc stability against S1 nuclease upon knockdown with FEN1 dsRNA, DNA2 dsRNA (C) and the double knockdown of FEN1 and DNA2 (F + D) (D). The presentation of the graph is the same as in Fig. 2B. Data represent the mean of 3 independent transfection experiments ± S.E.M. (C) and the mean of 2 independent transfection experiments with the bars representing the range of the values (D).
Bowmaker,
Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone.
2003, Pubmed
Bowmaker,
Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone.
2003,
Pubmed
Brewer,
The localization of replication origins on ARS plasmids in S. cerevisiae.
1987,
Pubmed
Brewer,
A replication fork barrier at the 3' end of yeast ribosomal RNA genes.
1988,
Pubmed
Brown,
Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism.
2005,
Pubmed
Brown,
Release of replication termination controls mitochondrial DNA copy number after depletion with 2',3'-dideoxycytidine.
2002,
Pubmed
Burgers,
Yeast exonuclease 5 is essential for mitochondrial genome maintenance.
2010,
Pubmed
Cerritelli,
Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice.
2003,
Pubmed
Cerritelli,
Ribonuclease H: the enzymes in eukaryotes.
2009,
Pubmed
Clayton,
Replication of animal mitochondrial DNA.
1982,
Pubmed
De,
A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability.
2007,
Pubmed
,
Xenbase
Duxin,
Human Dna2 is a nuclear and mitochondrial DNA maintenance protein.
2009,
Pubmed
Ellenberger,
Eukaryotic DNA ligases: structural and functional insights.
2008,
Pubmed
Gao,
DNA ligase III is critical for mtDNA integrity but not Xrcc1-mediated nuclear DNA repair.
2011,
Pubmed
,
Xenbase
Holt,
Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA.
2000,
Pubmed
Kaguni,
DNA polymerase gamma, the mitochondrial replicase.
2004,
Pubmed
Kang,
Dna2 on the road to Okazaki fragment processing and genome stability in eukaryotes.
2010,
Pubmed
Kao,
The protein components and mechanism of eukaryotic Okazaki fragment maturation.
2003,
Pubmed
Lakshmipathy,
The human DNA ligase III gene encodes nuclear and mitochondrial proteins.
1999,
Pubmed
,
Xenbase
Lakshmipathy,
Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity.
2001,
Pubmed
,
Xenbase
Lima,
Human RNase H1 discriminates between subtle variations in the structure of the heteroduplex substrate.
2007,
Pubmed
Liu,
Removal of oxidative DNA damage via FEN1-dependent long-patch base excision repair in human cell mitochondria.
2008,
Pubmed
Muylaert,
Knockdown of DNA ligase IV/XRCC4 by RNA interference inhibits herpes simplex virus type I DNA replication.
2007,
Pubmed
Pohjoismäki,
Human heart mitochondrial DNA is organized in complex catenated networks containing abundant four-way junctions and replication forks.
2009,
Pubmed
Pohjoismäki,
Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid.
2010,
Pubmed
Robberson,
Replication of mitochondrial DNA. Circular replicative intermediates in mouse L cells.
1972,
Pubmed
Ruhanen,
Mitochondrial single-stranded DNA binding protein is required for maintenance of mitochondrial DNA and 7S DNA but is not required for mitochondrial nucleoid organisation.
2010,
Pubmed
Simsek,
Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair.
2011,
Pubmed
,
Xenbase
Sunderland,
An evolutionarily conserved translation initiation mechanism regulates nuclear or mitochondrial targeting of DNA ligase 1 in Arabidopsis thaliana.
2006,
Pubmed
Timson,
DNA ligases in the repair and replication of DNA.
2000,
Pubmed
,
Xenbase
Wiegand,
Specificity of the S1 nuclease from Aspergillus oryzae.
1975,
Pubmed
Willer,
The yeast CDC9 gene encodes both a nuclear and a mitochondrial form of DNA ligase I.
1999,
Pubmed
Yang,
Biased incorporation of ribonucleotides on the mitochondrial L-strand accounts for apparent strand-asymmetric DNA replication.
2002,
Pubmed
Yasukawa,
Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand.
2006,
Pubmed
Yasukawa,
A bidirectional origin of replication maps to the major noncoding region of human mitochondrial DNA.
2005,
Pubmed
Zheng,
Human DNA2 is a mitochondrial nuclease/helicase for efficient processing of DNA replication and repair intermediates.
2008,
Pubmed
