ABSTRACT
Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle.
Subject(s)
Base Pairing/physiology , DNA Replication/genetics , DNA, Mitochondrial/metabolism , DNA, Single-Stranded/metabolism , RNA, Mitochondrial/physiology , Animals , DNA Helicases/genetics , DNA Helicases/metabolism , DNA, Mitochondrial/chemistry , DNA, Single-Stranded/chemistry , Gene Expression Regulation/physiology , Genomic Instability/genetics , HEK293 Cells , Humans , Mammals , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Mutagenesis, Site-Directed , Mutant Proteins/genetics , Mutant Proteins/metabolism , Nucleic Acid Conformation , RNA, Mitochondrial/chemistry , RNA, Mitochondrial/metabolismABSTRACT
Mitochondrial DNA is replicated by a unique enzymatic machinery, which is distinct from the replication apparatus used for copying the nuclear genome. We examine here the mechanisms of origin-specific initiation of lagging-strand DNA synthesis in human mitochondria. We demonstrate that the mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the light-strand origin of DNA replication (OriL). Using only purified POLRMT and DNA replication factors, we can faithfully reconstitute OriL-dependent initiation in vitro. Leading-strand DNA synthesis is initiated from the heavy-strand origin of DNA replication and passes OriL. The single-stranded OriL is exposed and adopts a stem-loop structure. At this stage, POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region. After about 25 nt, POLRMT is replaced by DNA polymerase gamma, and DNA synthesis commences. Our findings demonstrate that POLRMT can function as an origin-specific primase in mammalian mitochondria.
Subject(s)
DNA Replication , DNA, Mitochondrial/biosynthesis , DNA-Directed RNA Polymerases/physiology , DNA, Mitochondrial/chemistry , Gene Silencing , Humans , Models, Genetic , Nucleic Acid Conformation , Poly T/chemistry , Replication OriginABSTRACT
We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.
Subject(s)
DNA Replication , DNA, Mitochondrial/chemistry , Animals , DNA , Humans , Mammals , Nucleic Acid Conformation , Nucleic Acid Hybridization , RNAABSTRACT
In higher vertebrates, the DNA of mitochondria takes the form of circular molecules of approximately 16 kbp. These circles are arranged in multigenomic nucleoprotein complexes or nucleoids. It is envisaged that nucleoid superstructure makes a critical contribution to the twin processes of replication and segregation of mtDNA. Replication intermediates can be isolated from cells or solid tissues and separated on agarose gels in two dimensions to reveal a wealth of data on mechanisms of DNA replication. Using this technique we have demonstrated that many molecules of replicating mtDNA have extensive regions of RNA: DNA hybrid in higher vertebrates. More recently, we have extracted mitochondrial nucleoprotein and analyzed it by the same method to derive information on the distribution of DNA-binding proteins on mitochondrial DNA. Here we describe the procedures used to isolate intact mitochondrial replication intermediates from liver and cultured cells of higher vertebrates and the process of separating DNA fragments on neutral two-dimensional agarose gels.
Subject(s)
DNA, Mitochondrial/analysis , Electrophoresis, Gel, Two-Dimensional/methods , Mitochondria, Liver/chemistry , Animals , Blotting, Southern , Cells, Cultured , DNA Replication , DNA Restriction Enzymes , DNA, Mitochondrial/isolation & purification , Electrophoresis, Agar Gel , MiceABSTRACT
Expression of a proof-reading deficient form of mitochondrial DNA (mtDNA) polymerase gamma, POLG, causes early death accompanied by features of premature ageing in mouse. However, the mechanism of cellular senescence remains unresolved. In addition to high levels of point mutations of mtDNA, the POLG mutator mouse harbours linear mtDNAs. Using one- and two-dimensional agarose gel electrophoresis, we show that the linear mtDNAs derive from replication intermediates and are indicative of replication pausing and chromosomal breakage at the accompanying fragile sites. Replication fork arrest is not random but occurs at specific sites close to two cis-elements known as O(H) and O(L). Pausing at these sites may be enhanced in the case of exonuclease-deficient POLG owing to delayed resumption of DNA replication, or replisome instability. In either case, the mtDNA replication cycle is perturbed and this might explain the progeroid features of the POLG mutator mouse.
Subject(s)
Chromosome Breakage , Chromosome Fragile Sites , DNA Replication , DNA, Mitochondrial/biosynthesis , DNA-Directed DNA Polymerase/genetics , Animals , DNA Polymerase gamma , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/metabolism , DNA-Directed DNA Polymerase/metabolism , Female , Liver/metabolism , Mice , Mice, Mutant Strains , Mitochondria/enzymology , Progeria/genetics , Sequence Analysis, DNA , Single-Strand Specific DNA and RNA EndonucleasesABSTRACT
Using two-dimensional agarose gel electrophoresis, we show that mitochondrial DNA (mtDNA) replication of birds and mammals frequently entails ribonucleotide incorporation throughout the lagging strand (RITOLS). Based on a combination of two-dimensional agarose gel electrophoretic analysis and mapping of 5' ends of DNA, initiation of RITOLS replication occurs in the major non-coding region of vertebrate mtDNA and is effectively unidirectional. In some cases, conversion of nascent RNA strands to DNA starts at defined loci, the most prominent of which maps, in mammalian mtDNA, in the vicinity of the site known as the light-strand origin.
