Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells

Heini Ruhanen, Kathy Ushakov, Takehiro Yasukawa

Research output: Contribution to journalArticle

23 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2000-2007
Number of pages8
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1813
Issue number12
DOIs
Publication statusPublished - Dec 1 2011
Externally publishedYes

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DNA Replication
Mitochondrial DNA
Cultured Cells
Flap Endonucleases
DNA Ligase ATP
ribonuclease HI
DNA
Ribonucleases
Ligation
Mitochondria
Proteins
Okazaki fragments

All Science Journal Classification (ASJC) codes

  • Cell Biology
  • Molecular Biology

Cite this

Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells. / Ruhanen, Heini; Ushakov, Kathy; Yasukawa, Takehiro.

In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1813, No. 12, 01.12.2011, p. 2000-2007.

Research output: Contribution to journalArticle

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