Different models of replication

As the mitochondria originate from the symbiotic relationship of an early eukaryotic cell with a prokaryotic cell, the interesting question is whether the replication of the modern- day mitochondrial DNA resembles more that of its prokaryotic ancestor cell or that of eukaryotic cells. There are two competing models to describe mtDNA replication, the strand-displacement model and the conventional, coupled leading- and lagging-strand DNA synthesis model.

2.6.1.1 Strand-asynchronous replication of mtDNA

In 1972, studies by electron microscopy indicated that mammalian mtDNA is replicated in an unconventional way, the replicating mtDNA having a single-stranded branch (Robberson et al. 1972). This gave rise to the model presented by Clayton (1982), referred to as the strand-displacement model involving strand-asynchronous and asymmetric DNA replication. Until quite recently, mtDNA replication was thought to occur uniquely as described in this model.

In the strand-displacement model the mtDNA strands are replicated at different times as shown in Figures 2.6 and 2.8a. According to this model there are two replication initiation sites for the two strands, heavy (leading) and light (lagging) strands, and these initiation sites are located far apart from each other in the circular genome (Clayton 1982). Replication starts from the heavy strand replication (illustrated as green dotted line in Figure 2.6) initiation site, OH, and two thirds of the leading strand is replicated before

replication of the lagging strand from OL (illustrated as red dotted line in Figure 2.6), the

light strand replication initiation site, in the opposite direction is initiated. As the heavy strand is replicated the parental strands are separated from each other. Once the initiation site for light strand replication has been uncovered, lagging strand replication may begin.

All this involves continuous ssDNA synthesis taking places in both directions (Clayton 1982). O_H O_L O_H O_L O_H O_L O_L O_H O_L O_H O_H O_L O_H O_L O_H O_L O_H O_L O_H O_L O_H O_L O_L O_H O_L O_H O_L O_H O_L O_H

Figure 2.6. The strand-asynchronous replication model of the human mitochondrial DNA. Adapted from Clayton (2000).

2.6.1.2 Strand-synchronous replication of mtDNA

Holt et al. (2000) first presented evidence for a more conventional mode of mtDNA replication based on coupled leading- and lagging-strand DNA synthesis (see Figure 2.8b). This mechanism is similar to the one used to replicate mammalian nuclear DNA (Lodish et al. 1999). This model suggests that the two strands are replicated symmetrically and it occurs simultaneously. Only one replication initiation site is therefore needed and replication proceeds on both strands in the same direction at the same time. Bowmaker et al. (2003) reported that bidirectional replication starts downstream of OH that is the heavy strand replication origin. Yasukawa et al. (2005)

mapped two sites in the NCR for bidirectional replication initiation in cultured cells recovering from drug-induced mtDNA depletion. At OH one of the replication forks is

stalled whereas the other one continues replicating through the whole genome as shown below in Figure 2.7. The replication machinery replicates mtDNA only in the conventional 5’ - 3’ direction meaning that one of the strands is replicated in Okazaki fragments (see Figure 2.8b). Black arrows in Figure 2.7 denote the replication fork movement direction.

Holt et al. (2000) suggested that these two replication mechanisms might function in mammalian mtDNA under different conditions. Namely, the two replication mechanisms

introduced above are not mutually exclusive. In support of this idea, it was found that strand-coupled replication occurs after EtBr-induced mtDNA depletion when cells replicate mtDNA more actively. When the mtDNA is only maintained in cells that grow ‘normally’ replication seems to occur according to a mechanism more akin to the model presented by Clayton (1982). Twenty-four hours after mtDNA depletion, cells were found to contain replication intermediates from both replication modes indicating that the cells were shifting back to normal mtDNA maintenance from active amplification mode. Therefore, it is also possible to hypothesize that Clayton’s model with its single light strand replication initiation site is an extreme case of Holt’s model (Holt et al. 2000).

O_H O_H a b O_H a b O_H O_H a b O_H a b

Figure 2.7. Bidirectional strand-coupled replication of mtDNA. Adapted from Bowmaker et al. (2003) and Yasukawa et al. (2005).

2.6.1.3 RITOLS replication

There are many recent publications where authors using 2DNAGE have failed to detect the single stranded replication intermediates predicted by the Clayton model (Holt et al. 2000, Kajander et al. 2001, Yang et al. 2002, Bowmaker et al. 2003, Reyes et al. 2005, Yasukawa et al. 2005, Yasukawa et al. 2006). Instead of such partially single-stranded replication intermediates, two groups of double stranded replication intermediates were reported in these publications. The first are intermediates originating from coupled leading- and lagging-strand DNA synthesis (Holt et al. 2000, Kajander et al. 2001, Reyes

et al. 2005, Yasukawa et al. 2006). Secondly, Yang et al. (2002) established that the

incorporated on the light strand, which are finally converted to DNA (see Figure 2.8c). Yasukawa et al. (2006) subsequently found replication intermediates containing long RNA segments extending over the whole lagging-strand before lagging-strand DNA synthesis had taken place, introducing the concept of RITOLS replication i.e. RNA incorporation throughout the lagging strand.

These two groups of replication intermediates not only have different ribonucleotide content but also their initiation sites differ. Coupled leading- and lagging-strand DNA synthesis initiates from a broad zone of many kilobases (Bowmaker et al. 2003, Reyes et

al. 2005), whereas RITOLS replication initiates only within the NCR (Yasukawa et al.

2006). This finding indicates that replication might occur in several different ways in vertebrate mtDNA. On the other hand, the RITOLS type of replication resembles the strand-asynchronous model presented by Clayton (1982), since there is considerable delay between leading and lagging-strand DNA synthesis in both models. The single stranded replication intermediates visualized by means of electron microscopy in 1970´s and later by means of atomic force microscopy (Robberson et al. 1972, Kasamatsu and Vinograd 1973, Brown et al. 2005) may be explained on the basis of loss of ribonucleotide segments from RITOLS RIs.

Leading strand _{Leading strand}

Okazaki fragments 5´ ends of RNA Leading strand Lagging strand Template DNA Nascent DNA Nascent RNA a) Strand-displacement model

b) Coupled leading- and lagging strand synthesis

c) RITOLS replication

Leading strand _{Leading strand}

Okazaki fragments 5´ ends of RNA Leading strand Lagging strand Template DNA Nascent DNA Nascent RNA Leading strand

Leading strand _{Leading strand}

Okazaki fragments 5´ ends of RNA Leading strand Okazaki fragments 5´ ends of RNA Leading strand Lagging strand Leading strand Lagging strand Template DNA Nascent DNA Nascent RNA a) Strand-displacement model

b) Coupled leading- and lagging strand synthesis

c) RITOLS replication

Figure 2.8. Different modes of mtDNA replication. a) Strand-displacement model. b) Conventional coupled leading- and lagging-strand DNA synthesis. c) RITOLS replication. (Adapted from Holt 2009).