In Vivo Determination of Replication Origins of Human Mitochondrial DNA by Ligation-mediated Polymerase Chain Reaction

Abstract

A large part of replication is aborted in human mitochondria, the result being a D-loop. As few attempts have been made to distinguish free 5 ends of true replicate from those of abortive ones, we examined the 5 ends of true replicate of human mitochondrial DNA at one nucleotide resolution in vivo by making use of ligation-mediated polymerase chain reaction. The distribution and relative amounts of origins of the true replicate are exactly the same as those of total newly synthesized heavy strands, which means that the abortion of replication is independent of 5 ends. Treatment of DNA with RNase H frees 5 ends on both heavy and light strands. This is the first in vivo evidence for covalently attached primer RNA to nascent strand in human mitochondrial DNA.Replication of mitochondrial DNA (mtDNA) begins with synthesis of the heavy strand (H strand) 1 from the replication origin O H , following transcription of the light strand (L strand). Transcription of the L strand is regulated by the L strand promoter (LSP) sequence. When synthesis of the H strand has proceeded about two-thirds, synthesis of the L strand begins from replication origin O L . Thus, the total replication rate of mtDNA is determined by H strand synthesis (for reviews, see Refs. 1-3). The multiple replication origins of human mitochondrial H and L strands have been determined as free 5Ј ends of mtDNA extracted from prepared mitochondria. The molecular mechanism and physiological significance of these events are poorly understood.A large part of the synthesis of H strand is aborted, leaving a displacement loop ((D-loop) or 7 S DNA). For example, over 95% of the newly synthesized H strand is a D-loop in the case of mouse L cells (4). This makes it difficult to precisely determine origins of the true replication form (nascent H strand) (5). Few investigators have distinguished the free 5Ј end of the true replicate form from that of the D-loop even though selective detection of the free 5Ј end of the true replicate form is critical to determine replication origin. Although the 5Ј ends of the nascent H strand are suggested to be the same as those of D-loop in human mitochondria (5), it is uncertain whether the distribution and relative amount of the 5Ј ends of nascent H strand are exactly the same as those of the D-loop. In addition, the free 5Ј ends of human mtDNA are not set at one nucleotide resolution (6). To precisely determine the replication origin but not the simple free 5Ј end, it is required to selectively detect the replication origin at a higher resolution.We made use of the ligation-mediated polymerase chain reaction (LMPCR) to detect free 5Ј ends (7,8), an approach which makes feasible use of total DNA extracted directly from whole cells. By selective amplification of nascent H (not D-loop), we determined precise and comprehensive replication origins of the H strand in vivo. Here, we describe sites of free 5Ј ends and the transition sites of RNA to DNA for both H and L strands at one nucleotide resolution.

Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria

“…Fractionation by sodium-borate gelelectrophoresis resolved four 7S DNA species of ∼560-650 nt, which are concordant with those defined previously (8) with free 5′ ends mapping to np 111, 150, 168, and 191 (23). Treatment with RNase HI did not alter their lengths (Fig.…”

Replication Origin of Mitochondrial DNA in Insects

“…Ligation-mediated PCR: LMPCR was performed according to previously described methods (Mueller et al 1992;Kang et al 1997). The primers used in this study are shown in Figure 1.…”

“…These are located around the O R and are widely conserved among vertebrate species. Immediately upstream of the O R of the H-strand, conserved sequence blocks (CSBs) are present and are suggested to be responsible for the initiation of the H-strand replication (Tapper and Clayton 1981;Cairns and Bogenhagen 1986;King and Low 1987;Kang et al 1997). CSBs are thought to be involved in generating the 39 ends of the RNA primers, which are required for the DNA synthesis of the H-strand (Shadel and Clayton 1997;Taanman 1999).…”

“…Since replication of Drosophila mtDNA proceeds unidirectionally from the O R Wolstenholme 1978, 1980), nascent DNA strands possess the free 59 ends at the O R . Therefore we determined the position of the free 59 ends of the DNA strand at 1-nucleotide resolution within the A 1 T-rich region for both strands, using the ligation-mediated PCR (LMPCR) method (Mueller et al 1992;Kang et al 1997). Then, the nucleotide sequences around the O R were compared among the four Drosophila species to identify potential regulatory sequences that may be involved in the replication initiation process.…”

Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro

“…DNA synthesis from O H is unidirectional and proceeds to displace the parental heavy strand (H-strand). After leading-strand synthesis has reached two-thirds of the genome, it activates O L and lagging-strand DNA synthesis then initiates in the opposite direction (2)(3)(4). Recently, analysis of mtDNA replication intermediates by atomic force microscopy revealed that the strand-displacement model is an oversimplification and that additional initiation sites for lagging-strand mtDNA synthesis exist on the light strand (L-strand) (5).…”