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Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells
- Source :
- Biochimica et Biophysica Acta
- Publisher :
- Elsevier B.V.
-
Abstract
- 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.<br />Highlights ► The data suggest that DNA ligase III is the replicative DNA ligase in human mitochondria. ► RNase H1 is indispensable for Okazaki fragment processing in human mtDNA replication. ► On the other hand, knockdown of neither DNA2 nor FEN1 affected mtDNA replication. ► Mitochondria utilise distinct proteins for Okazaki fragment maturation from nucleus.
- Subjects :
- DNA Ligases
Flap Endonucleases
Blotting, Western
Ribonuclease H
Eukaryotic DNA replication
Bone Neoplasms
Xenopus Proteins
DNA replication
Primosome
DNA, Mitochondrial
Thymidine Kinase
Article
Okazaki fragment
DNA Ligase ATP
Tumor Cells, Cultured
Humans
Mitochondrion
Poly-ADP-Ribose Binding Proteins
Molecular Biology
Osteosarcoma
biology
Okazaki fragments
Cell Biology
DNA
Molecular biology
Mitochondrial DNA
Mitochondrial DNA replication factor
Blotting, Southern
Prokaryotic DNA replication
biology.protein
DNA polymerase I
Primer (molecular biology)
Mitochondrial DNA replication
Subjects
Details
- Language :
- English
- ISSN :
- 01674889
- Issue :
- 12
- Database :
- OpenAIRE
- Journal :
- Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
- Accession number :
- edsair.doi.dedup.....daf1144ea2c80543234c1ace9e8bcc40
- Full Text :
- https://doi.org/10.1016/j.bbamcr.2011.08.008