The role of meiotic homologous recombination, either crossing-over or gene conversion, is the acquisition of genetic diversity in each species by reorganizing parental alleles, to facilitate adaptation to environmental changes. In meiosis, the general process to produce haploid cells from diploid mother cells, chromosome duplication is followed by two rounds of cell division without DNA replication. Gene conversion and crossing-over are induced at very high frequencies in the early phase of the first meiotic cell division, by site-and timing-specific double-stranded breakage catalyzed by SPO11. Crossing-over is essential for the precise sorting of each of the homologous chromosome pairs to the opposite poles in the first meiotic cell division, to accomplish the meiotic segregation of homologous chromosomes. This is supported by the observations that most meiotic recombination-deficient mutants cause meiotic nondisjunction, to produce aneuploids. In human, reduced meiotic recombination induces nondisjunction of chromosome 21, which causes Down Syndrome [24].

Meiotic crossing-over is strictly restricted between alleles, and generates various new combinations of alleles derived from both parents to increase genetic diversity. Crossing-over between nonallelic genes necessarily results in chromosomal aberrations, but gene conversion occurs between not only alleles but also non-allelic genes with similar sequences (homologous recombination), and consequently, occasionally produces chimeric genes with additional genetic variations. Both meiotic crossing-over and gene conversion depend on RecA-family proteins, Rad51 and Dmc1, as well as a recombination mediator (Rad52 in S. cerevisiae).

Somatic gene conversion among the members of a DNA sequence family with some sequence variations results in extensive sequence diversification. An example is the production of genes encoding immunoglobulins with various antigen-specificities in a chicken cell line (DT40), derived from bursa B cells [25–27]. This gene conversion allows a 4–20% level of mismatched base-pairs between the donor and recipient DNA sequences [28], which is orders of magnitude higher than the levels tolerated by meiotic gene conversion and double-stranded break-repair observed in somatic cells. The hypervariable region is also a hotspot of somatic hypermutation, and may contribute to immunoglobulin diversification. When DT40 cells were treated with trichostatin A, a histone deacetylase inhibitor, almost all of the cells within the culture recombined at the loci under the optimal conditions, and the induced recombination was suppressed simply by removing the drug. This finding enabled the development of a quick method to generate clones of functional antibodies (ADLib system) [29]. DNA sequence analyses of these established cell lines revealed that the genes encoding the newly obtained, functional antibodies were mostly generated by gene conversion, and the contribution of somatic hypermutation was very limited, even though the trichostatin A-treatment also enhanced somatic hypermutation [29]. Details about the rapid generation of specific antibodies by the ADLib system are described in the review by Kunihiro Ohta, in this issue. The results obtained with the ADLib system suggest that gene conversion between similar sequences of a DNA sequence family has significant power to diversify genes and even to create genes encoding proteins with new functions, as discussed previously for DNA shuffling [30].

Each cell contains a number of mtDNA copies, which suffer from a high frequency of mutagenesis and are recombined to generate either gene conversion products or crossing-over products in yeast and other organisms [10]. Thus, it might be expected that the sequence of mtDNA would be extensively diversified, but as mentioned, mtDNA maintains homoplasmy. This suggests that mitochondria have potent mechanisms to suppress the diversification of mtDNA and to maintain mtDNA homoplasmy.

Occasionally, repair synthesis that starts at a D-loop continues without capturing the second end, with associated lagging strand DNA synthesis, as first shown in bacteriophage T4 [31], bacteria [32,33] and yeast [34]. This prolonged DNA synthesis (double-stranded break induced DNA-replication, often abbreviated as BIR) in yeast was shown to cause coconversion to the donor genotype (Figure 2) [35,36]. Double-stranded break-induced DNA replication occurs when the second end is not available, as in the case of a collapsed replication fork, which is recovered by this double-stranded break-induced DNA replication-dependent mechanism [37]. When the second end was available in S. cerevisiae, most of the double-stranded breaks induced in diploid wild-type cells were repaired by Rad51-dependent gene conversion, but when the double-stranded breaks were induced in diploid rad51Δ/ad51Δ cells, one-third of the breaks were repaired by double-stranded break-induced DNA replication, while none was repaired by simple gene conversion. This double-stranded break-induced DNA replication mechanism absolutely depends on Rad52, but does not require Rad51 [34].

MtDNA recombination, involving crossing-over and gene conversion, has been intensively studied and well characterized in the yeast Saccharomyces cerevisiae [10]. Some sequence-specific endonucleases, such as Endo.SceI [38,39] and I-SceI [40,41], were shown to induce very efficient gene conversion at their cleavage sites in mtDNA, and the cleaved DNA always acted as the recipient, as also observed in nuclear gene conversion. Endo.SceI is a heterodimer of Ens2, encoded by a mitochondrial gene, and a nuclear chromosome-encoded mitochondrial 70 kDa heat shock protein (mtHSP70) [42], and numerous cleavage sites for Endo.SceI exist in the yeast mtDNA [39]. On the other hand, I-SceI and other mitochondrial sequence-specific endonucleases (homing endonucleases) are homodimers encoded within mitochondrial introns, functioning in the transposition of the introns into their intronless alleles (homing) through gene conversion, and each has a rare cleavage site within its intronless allele [40]. The sequence-specific endonuclease-induced gene conversion induced by I-SceI is the most efficient; i.e., all progeny of the mating of I-SceI intron-plus cells and I-SceI intronless cells are I-SceI intron-plus. Note that I-SceI is the name of an intron of the 21S ribosomal RNA gene as well as the name of the sequence-specific endonuclease encoded in the intron [43], and that I-SceI and Endo.SceI [38] are different endonucleases with diverse target specificities.

Until the mid-nineties, the elucidation of the molecular mechanisms of homologous mtDNA recombination had been hampered by unsuccessful attempts to isolate mtDNA recombination-deficient mutants, until the identification of the first defective yeast mutant, mhr1-1. By using the efficient mtDNA gene conversion induced by I-SceI or Endo.SceI and developing a “mitochondrial crossing in haploid” screening system, a homologous mtDNA recombination-defective mutant was isolated for the first time [11]. The mutation is a recessive, single base-substitution in a nuclear gene, MHR1, and it generates an amino acid-substitution in the mitochondrial protein, Mhr1 [44].

The mhr1-1 mutation largely suppresses the gene conversion induced by I-SceI and Endo.SceI [11]. Although only a mild reduction in mtDNA crossing-over was observed in mhr1-1 cells [11], this is due to the crossing-over between unlinked mtDNA markers, and subsequent physical tests revealed that mhr1-1 clearly decreased mtDNA crossing-over [45].

Before mhr1-1 was found, two genes or proteins that may be related to mtDNA recombination were known. Pif1 is a DNA helicase [46], and its mutant was isolated by its effect on recombination between a specified type of ρ⁻ DNA (petite; respiration defective mtDNA with a large deletion) and ρ⁺ (respiration proficient) mtDNA, but this mutation does not affect recombination between ρ⁺ mtDNAs [47,48]. Pif1 also functions in nuclei at telomere [49] and minisatellite DNA [50]. Cce1 encodes a DNA endonuclease that resolves the Holliday junction in vitro, but a recombination-deficient phenotype was not reported [51,52].

MHR1 encodes a protein that catalyzes D-loop formation from homologous double-stranded DNA and linear single-stranded DNA in the absence of ATP. The mhr1-1 mutation is a single amino acid-substitution causing the inactivation of the D-loop forming activity of Mhr1 [12]. Thus, the D-loop forming activity of Mhr1 explains the deficiencies of mhr1-1 in homologous mtDNA recombination, either gene conversion or crossing-over. The mhr1-1 mutation generates a pleiotropic phenotype in ρ⁺ cells. First, it showed defective mtDNA maintenance at a higher temperature, with associated defects in replicated mtDNA transmission into daughter cells (temperature sensitive partitioning) [11,12]. Second, mhr1-1 exhibited slower segregation of heteroplasmic cells to generate homoplasmic cells at a sub-lethal temperature, and the overexpression of Mhr1 in ρ⁺ cells accelerated the segregation of heteroplasmic cells, indicating that Mhr1 plays an important role in mtDNA homogenization to maintain homoplasmy [13].

The mechanistic roles of Mhr1 in mtDNA partitioning into daughter cells were revealed when the ρ⁺ mtDNA (ca. 80 kbp) was analyzed in mother cells and growing buds separated from dividing cells, by the use of pulsed-field gel electrophoresis. The major species of mtDNA in the mother cells was multimers, as observed in whole cells, but in the bud, the major mtDNA species was monomers [12]. Detailed information about the forms of mtDNA in the mother and daughter cells was obtained by the analysis of small ρ⁻ mtDNA (ca. 10 kbp) by 1D and 2D agarose gel-electrophoresis. The major species of mtDNA in cells are linear head-to-tail multimers of varying sizes (concatemers) in the mother cells, and circular monomers in daughter cells [12,13]. The amounts of ρ⁻ mtDNA concatemers depend on the activity of Mhr1 within cells, as revealed by the effects of mhr1-1 and the overexpression of Mhr1 in yeast cells [13]. These features are very similar to those observed in the late phase of DNA replication and packaging of E. coli phages lambda and E. coli phage T4 DNA replication; i.e., recombination-dependent formation of concatemers, and selective packing of concatemers into phage capsids by terminase [31,53]. The defective partitioning of mtDNA in mhr1-1 yeast cells indicates that the concatemers formed by Mhr1-dependent mtDNA replication are an essential intermediate for partitioning promoted by the terminase-like enzyme (Figure 3).

Concatemers are formed from circular monomers of DNA, by either crossing-over type homologous recombination or rolling circle DNA replication. A series of pulse labeling and chase experiments of ρ⁻ mtDNA in synchronized cells supported rolling circle DNA replication and excluded crossing-over as the major pathway of concatemer formation in the mother cells, and also showed that the concatemers in the mother cells are the precursors of the circular mtDNA monomers in the growing buds [13]. These results indicated that rolling circle DNA replication is the major pathway to form ρ⁻ mtDNA concatemers, which are selectively transmitted into buds [12,13]. Thus, yeast cells have the Mhr1-dependent mechanism to replicate mtDNA in the rolling circle mode, to form concatemers and to transmit them selectively into daughter cells (mtDNA partitioning). These Mhr1-dependent mechanisms provide an explanation for the homogenization of heteroplasmic ρ⁺ mtDNA to generate homoplasmic ρ⁺ cells (Figure 3): Mhr1-dependent rolling circle replication allows the progeny of a template mtDNA molecule to dominate in a multicopy mtDNA population within a cell, and consequently, heteroplasmic mtDNA is rapidly homogenized during mitotic yeast cell growth to generate homoplasmic cells. In addition, the selective transmission of concatemers, the products of rolling circle replication, to daughter cells further enhances mtDNA homogenization [13].

In S cerevisiae ρ⁻ mitochondria, rolling circle DNA replication is initiated by a double-stranded break introduced at a mtDNA replication origin, such as ori5 [45]. The composition of the yeast mtDNA replication origin resembles that of the human heavy chain replication origin [55,56]; i.e., the sequence overlaps a transcription promoter and is very AT-rich, with three GC-rich clusters. The yeast hypersuppressive ρ⁻ mtDNA has been used as an excellent model system for studying the mechanisms of mtDNA replication and its initiation. Hypersuppressive ρ⁻ mtDNA is a small fragment (1 kbp or less) of ρ⁺ mtDNA (ca. 80 kbp) that contains an active mtDNA replication origin, and it has an extreme selective advantage over ρ⁺ mtDNA or neutral ρ⁻ mtDNA. Among the yeast mtDNA replication origins, ori5 is the most active [55,57]. The selective replication and propagation of the hypersuppressive ρ⁻ mtDNA over ρ⁺ mtDNA or neutral ρ⁻ mtDNA depends on Mhr1 [45], but does not require the transcriptional RNA polymerase (Rpo41) of mitochondria [58]. These observations provide additional support for Mhr1-initiated rolling circle replication, but not for RNA-primed replication, as the major mechanism for yeast mtDNA replication. Rolling circle replication should be associated with lagging strand synthesis, and a primase has not been found in any mitochondria. This observation also suggests that Rpo41 does not act as a primase for lagging strand synthesis.

A series of biochemical and genetic analyses of a hypersuppressive ρ⁻ mtDNA containing ori5 revealed that Ntg1 by itself introduced a double-stranded break in ori5, in mtDNA isolated from oxidatively stressed yeast cells in vitro. In addition, the double-stranded break introduced by Ntg1 in vivo is responsible for the initiation of rolling circle DNA replication, the selective replication and propagation of hypersuppressive ρ⁻ mtDNA, and crossing-over in yeast mitochondria [45]. These findings are somewhat surprising, since Ntg1, a homologue of E. coli endonuclease III, is a well-characterized base-excision repair enzyme that introduces a single-stranded break at nucleotides with an oxidatively damaged base, by its DNA-N-glycosylase- and AP-site-specific DNA-lyase activities [59]. The ability of Ntg1 to cause double-stranded breaks was a novel finding. The Ntg1 treatment of plasmid DNA isolated from heavily oxidatively stressed E. coli cells, under the same conditions, used for the detection of a double-stranded break at ori5, generated only single-stranded breaks, but no double-stranded breaks [45]. These results suggest that mtDNA has a specific structure at the mtDNA replication origin, ori5, for the regulation of mtDNA replication initiation under oxidatively stressful conditions.

MtDNA replication is not coupled with the cell division cycle. The mtDNA copy number changes in response to physiological conditions. Oxidative stress is known to increase the mtDNA copy number in mammals. However, the mechanisms regulating mtDNA replication and copy number are not known [60,61]. The requirement for Ntg1 in the initiation of rolling circle DNA replication suggests that reactive oxygen species (ROS) act directly as signaling mediators to regulate the yeast mtDNA replication. This assumption was confirmed by studies using isolated mitochondria and intact cells with the hypersuppressive ρ⁻ mtDNA. When an appropriate type of oxidative stress was applied to isolated mitochondria, the levels of the ori5-specific double-stranded break and the copy number of the hypersuppressive ρ⁻ mtDNA simultaneously increased [45,62]. Since isolated mitochondria were used in these studies, the effects of nuclear gene expression and metabolic systems could be ignored. Ntg1 is able to recognize an oxidized base on single-stranded DNA and to cleave the DNA at the site, and the mtDNA replication origin (ori5) has a local single-stranded structure in vivo, as shown by the locus-specific sensitivity to the single-stranded DNA- specific S1 nuclease and resistance to double-stranded DNA-specific restriction endonucleases [62].

Since single-stranded DNA is much more sensitive to damaging agents, including ROS, than double-stranded DNA, the local single-stranded nature and the enzymatic specificities of Ntg1 explain the locus-specific oxidation of bases and the double-stranded breakage at ori5 [62] (Figure 4). Thus, ROS act as signal mediators to regulate mtDNA replication and copy number [62].

The activity of the TCA cycle (tricarboxylic acid cycle, citric acid cycle) is regulated at the transcriptional level by nutritional and other environmental conditions [63]. The TCA cycle itself is a source of ROS production at the stage of α-ketoglutarate dehydrogenase, especially when the NADH/NAD⁺ ratio is increased [64]. Although the role of the mtDNA copy number in the regulation of mitochondrial gene expression has not been clarified, this, and the function of ROS as a mediator of mtDNA copy number control, suggest an interesting possibility: i.e., the activation of the TCA cycle to produce NADH, enhances ROS production and triggers an increase in the mtDNA copy number, which in turn might increase the expression of genes encoded in the mitochondrial genome.

It is well established that yeast ρ⁺ mtDNA replicates by a Mhr1-mediated recombination-dependent mechanism, and that ρ⁻ mtDNA replicates by a rolling circle mode in S. cerevisiae, as described. Concatemers are the predominant form of ρ⁺ mtDNA in Saccharomyces yeast [65–67]. However, whether ρ⁺ mtDNA replicates by the rolling circle mode or not in yeasts is under debate due to a lack of direct evidence. Historically, mtDNA replication is initiated by RNA synthesis by a transcriptional RNA polymerase (Rpo41 in S. cerevisiae). Although the maintenance of ρ⁺ mtDNA requires Rpo41, Rpo41 is not required for mtDNA replication origin (including ori5) functions, as described above [58].

When concatemers are formed through multiple rounds of crossing-over from circular or linear monomeric DNA, DNA networks are formed during the process. S. cerevisiae has branched mtDNA molecules [67]. Holliday junctions are obligatory intermediates of crossing-over, and a Holliday junction resolvase is required to finish concatemer formation. A typical example is E. coli phage T4 DNA replication, in which branched molecules or networks of DNA are formed from permutated linear DNA molecules, and Endo VII, which resolves the Holliday junctions, is required before packaging by terminase [31]. On the other hand, S. cerevisiae ρ⁺ mtDNA maintenance does not depend on Cce1, the sole yeast mitochondrial Holliday junction resolvase [51,52,68], and thus, the mtDNA network detected in yeast mitochondria is not an essential intermediate of ρ⁺ mtDNA replication and partitioning in S. cerevisiae. Since crossing-over and rolling circle replication are the alternative pathways to form concatemers from monomeric mtDNA (the major form of mtDNA in daughter cells), this supports rolling circle replication as the major pathway of ρ⁺ mtDNA inheritance. The concatemer formation by Cce1-dependent crossing-over appears to act as a minor pathway for the maintenance of ρ⁻ mtDNA, because mhr1-1 cells maintain a very small amount of ρ⁻ mtDNA at the restrictive temperature, and the presence of the cce1 null mutation in the cell is required for the complete removal of mtDNA [12].

The copy number of ρ⁺ mtDNA is increased in isolated yeast mitochondria by a treatment with limited amounts of hydrogen peroxide, and this increase depends on both Mhr1 and Ntg1, as in the case of hypersuppresive ρ⁻ mtDNA [62]. The copy number of ρ⁺ mtDNA in yeast cells increased when the cells were cultured under conditions that increased intracellular ROS [62]. These results also support the proposal that ρ⁺ mtDNA replicates by the recombination-dependent mechanism, including Mhr1 and Ntg1. In addition, in the ROS-induced increase in mtDNA in ρ⁺ cells, the copy numbers of genetic markers on mtDNA distant from ori5 or other active replication origins changed simultaneously, without a detectable difference from that of ori5 [62]. This observation supports rolling circle replication and is inconsistent with DNA replication initiated at each mtDNA genomic unit (replicon), since, in the latter mode, the copy number of ori5 should increase before those of the genetic markers distant from a replication origin, just after the induction of mtDNA replication by ROS.

The heteroplasmy of even neutral mitochondrial genetic markers segregates within a few generations into homoplasmy in mammals [87–89]. In the traditional theory of mammalian or human mtDNA replication, at the heavy chain replication origin, RNA is synthesized by the mitochondrial RNA polymerase for transcription, processed by an endonuclease, and then heavy chain synthesis is initiated at the 3′ end of the RNA, as a primer. The heavy chain synthesis continues to the light chain replication origin associated with D-loop formation (a different D-loop than that formed by homologous pairing in recombination), and then pauses. The light chain is synthesized along the D-loop, and then the entire mtDNA is replicated [90–92]. This traditional theory was challenged by the coupled leading- and lagging-strand synthesis mechanism [93–96]. Both theories explain how a circular mtDNA monomer replicates into two circular sister monomers. However, if each mtDNA copy is duplicated independently, followed by random 34 upon cell division, then the segregation of heteroplasmic parental calls, containing hundreds to thousands of mtDNA copies, into homoplasmic progeny is difficult to explain. Therefore, extensive reduction of the mtDNA copy number may occur at a critical step for the segregation (genetic bottleneck). The existence of this genetic bottleneck is under debate, and no gene function at the bottleneck step was found in mammals [4–7].

It was once believed that mitochondrial DNA (mtDNA) does not recombine in mammals, but recently various results supporting homologous mtDNA recombination in human and mammals have been published [97–99]. Although it was only shown in ρ⁻ yeast, rolling circle replication followed by selective transmission of the replication products, concatemers, to the progeny is the sole mechanistic model with genetic and biochemical support.

In yeast mitochondria, the mtDNA can be regarded as tandem DNA repeats (i.e., concatemers) of mitochondrial genome units. The nuclear genome also has various tandem DNA repeats (repeated DNA sequences or repetitive elements), including rDNAs, LINEs, SINEs and satellite DNAs. As in mitochondrial DNA, it has been recognized that each of these repeated DNA sequences consists of a homogeneous sequence, and the homogenization of the sequences has been discussed, in terms of concerted evolution [100]. Unequal crossing-over and gene conversion have been considered as mechanisms for concerted evolution. A recent study of hybrid scallops revealed that the very rapid homogenization of rDNA sequences into the maternal genotype is likely to occur by biased mitotic gene conversion [101]. Eukaryotic cells generally contain extrachromosomal circular DNA with heterogeneous sizes, consisting of sequences of repeated chromosomal DNA sequences. The findings in human cells of circular multimers of rDNA and of structures corresponding to intermediates of rolling circle DNA replication suggest a mechanism for the concerted evolution or homogenization of tandem repeats [102]. Telomeres consist of repeated sequences, and in the absence of telomerase, the telomere is maintained by Rad52-dependent and Rad51-independent rolling circle replication in yeast [36]. Therefore, it would be interesting to consider the possibility that cells use rolling circle DNA replication as a mechanism to homogenize (or homoplasmy) repeated DNA sequences, in both the nuclei and mitochondria of various eukaryotes.