Models of mtDNA replication

Three different models describe the mechanisms of mitochondrial replication: (1) The strand coupled DNA replication model, (2) the strand displacement model (SDM) and (3) the ribonucleotide incorporation throughout the lagging strand (RITOLS) model (Clayton et al., 1991; Holt et al., 2000; Yang et al., 2002).

(1) Strand coupled replication is similar to the classical replication model of the nucleus with a leading and lagging strand. Replication was proposed to be unidirectional, starting at several origins of replications (Bowmaker et al., 2003). Using 2D agarose gel electrophoresis (2D-AGE), replication intermediates fitting to that model could be observed. However, these intermediates represent only a minority, relative to intermediates fitting to RITOLS or SDM (see below) (Holt et al., 2000; Holt et al., 2012).

Introduction

21 In support with strand coupled replication, the whole machinery for maturation of Okazaki fragments can be found in mitochondria (Futami et al., 2007; Liu et al., 2008;

Duxin et al., 2009; Holt et al., 2009). However, early EM images of mouse mtDNA support a different model in which replication occurs not simultaneously on both strands (Kasamatsu and Vinograd, 1972). This observation can be explained with both, the RITOLS and SDM, but not with strand coupled replication.

(2) SDM and (3) RITOLS have in common that replication of the heavy and light strand is uncoupled and do not involve a lagging strand or Okazaki fragments (Fig 1.10 A, Berk et al., 1974; Berk and Clayton, 1976). After initiation of replication at OH, the daughter H-strand is synthesized by POLγ in 5’-3’-direction. After POLγ has synthesized two thirds of the new H-strand, it reaches the OL. The origin of light strand replication is located in a cluster of five tRNAs and is exposed in its single stranded conformation by the elongation complex. Upon exposure, OL forms a loop structure which triggers POLRMT to initiate the synthesis of a 25 nt primer. This primer works as substrate for POLγ to initiate L-strand replication in 5’-3’-direction. TWINKLE is not necessary for L-L-strand synthesis since it is already unwinded and single stranded. Finally, H- and L-strand replication proceeds continuously until both reach a full circle (Clayton et al., 1991; Wanrooij et al., 2012 (1)). In this model it is very important to explain how the displaced H-strand is stabilized, avoiding the exposure of direct repeats which can cause deletions, at least in bacterial plasmids (Born et al., 1991). RITOLS and SDM differ in the mechanism which stabilizes the single stranded displaced strand during synthesis of the daughter H-strand.

According to RITOLS the displaced H-strand is stabilized by processed RNAs. This model is also based on 2D-AGE analysis in combination with the use of endonucleases. This study together with analysis of its mass show that the displaced H-strand is double stranded (Yasukawa et al., 2006). Since the displaced H-strand cannot be cut by endonucleases, it can be ruled out that the H-strand is dsDNA. In contrast, RNAse H is able to cut DNA:RNA hybrids and removes the double stranded parts of the H-strand, revealing that the DNA is covered with RNA (Yang et al., 2002). Studies show that stabilizing RNA-fragments have a length of about 200-600 nt. However, the source of this RNA is not clarified. Most likely the RNA is derived from preformed and matured transcripts which are hybridized with the displaced strand, termed as the “bootlace model” (Yasukawa et al., 2006). Critics of RITOLS argue that the proteins involved in that hybridization process are not identified so far and that it is unclear how highly structured tRNA and rRNA can be melted to enable DNA binding (Holt et al., 2014).

Introduction

22 The SDM prefers mtSSBP as the important molecule stabilizing the displaced H-strand during replication. Chromatin immunoprecipitation (ChIP) experiments show that mtSSBP cover the displaced strand and show exclusive affinity to the H-stand and nearly no binding at the L-strand. Furthermore, mtSSBP shows its strongest density near the OH and is decreasing towards the OL and another strong peak upstream of OL. This fits to SDM since the areas in which mtDNA will be present as ssDNA for a longer period show higher levels of mtSSBP (Fuste et al., 2014).

Figure 1.10 Replication of mtDNA: A) Principle of SDM and RITOLS: Replication of mtDNA is initiated at OH within the NCR and the daughter H-strand is synthesized by the action of POLγ and TWINKLE. The parental H-strand is displaced, remains single stranded and is stabilized by either RNA (RITOLS, cyan) or mtSSBP (SDM, orange). When the replication machinery has synthesized two thirds of the new H-strand, OL is exposed and L-strand synthesis is initiated. Since the parental H-strand is already single stranded, no TWINKLE is necessary. Both replication machineries continue till mtDNA is replicated. B) D-loop formation: Synthesis of the daughter H-strand can be terminated after 650 nt at the TAS region, resulting in the 7S DNA. The D-loop is formed as the 7S DNA remains at the mtDNA and the parental H-strand remains displaced. Depending on the organism and cell type the D-loop has a specific lifetime after which the 7S DNA dissociates from the mtDNA.

Studies favoring RITOLS and SDM show strong evidences that the displaced H-strand is in most cases covered with protein or RNA, but not undergoing replication of a classical lagging strand including the formation of Okazaki fragments. There is no consensus if mtDNA replication follows RITOLS or SDS, since there is strong evidence for RNA and

Introduction

23 mtSSBP to cover the displaced lagging strand (Yasukawa et al., 2006; Fuste et al., 2014).

It has been suggested that both RNA and mtSSBP are involved in this process. Studies show that at least 80% of the displaced DNA strand is covered with RNA and single stranded stretches are not longer than 100-200 nt. These gaps could be filled with mtSSBP (Pohjoismaki et al., 2010; Wanrooij and Falkenberg, 2010).

Some scientists claim that replication of mtDNA may not only have a single mechanism or even a single origin of replication, but different mechanisms initiated at different positions. An indication for that could be the presence of several mitochondrial DNA polymerases found in Trypanosomes which could be required for different replication mechanisms. Also in humans, a second polymerase was found to be active in mitochondria: PrimPol. Multiple polymerase would provide another degree of freedom to regulate mitochondrial replication and thereby there might not exist only one single mode of mtDNA replication (Klingbeil et al., 2002; Holt et al., 2012; Garcia-Gomez et al., 2013). However, all data suggest the majority if replication occurs with an displaced strand that is covered with RNAs and mtSSBP, as well (Yasukawa et al., 2006; Wanrooij and Falkenberg, 2010; Holt et al., 2012, Fuste et al., 2014)