When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination. Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate homologous recombination and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast1 (MOC1) as a plastid DNA HJ resolvase in algae and plants. Although Cruciform cutting endonuclease1 (CCE1) was identified as a mitochondrial DNA HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrate that MOC1 depletion in the green alga
Chlamydomonas reinhardtii and the moss
Physcomitrella patens induced ectopic recombination between short dispersed repeats in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss
P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of plastid DNA, MOC1 depletion in
P. patens induced short dispersed repeat-mediated ectopic recombination in mitochondrial DNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (
RECA2) and mitochondrion-targeted (
RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants as well as in plastids in algae and plants.Mitochondria and plastids were established in eukaryotic cells by endosymbiotic events of α-proteobacterial and cyanobacterial ancestors, respectively (
Gray, 1992;
Archibald, 2015). Reminiscent of their bacterial ancestors, both organelles possess their own genomes and proliferate by division of preexisting ones (
Martin and Kowallik, 1999). Plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) encode some components of the photosynthetic apparatus and respiratory chain, respectively (
Allen, 2003). Thus, to maintain the functions of plastids and mitochondria, ptDNA and mtDNA must faithfully replicate and segregate during proliferation of the organelles.The mitochondrion and plastid possess multiple copies of DNA, which are organized with proteins into nucleoids (
Kuroiwa, 1991;
Pfalz and Pfannschmidt, 2013). Nucleoids, which can be visualized as dot-like or globular structures in mitochondria and plastids when stained with DNA-specific fluorochromes such as 4′, 6-diamidino-2-phenylindole (DAPI) or SYBR Green I, are ubiquitously observed in diverse lineages of algae and plants (
Kuroiwa, 1991;
Sato, 2001;
Kobayashi et al., 2016). The morphology of nucleoids dynamically changes according to cell cycle progression and development (
Powikrowska et al., 2014). For example, in the unicellular green alga
Chlamydomonas reinhardtii, ∼80 copies of ptDNA are packaged into 5 to 8 globular nucleoids in a single cup-shaped plastid during the gap 1 (G1) phase (
Armbrust, 1998). Prior to plastid division, during the synthesis (S) and mitosis (M) phases, plastid nucleoids change into filamentous structures and are scattered throughout the plastids. Then the nucleoids are inherited by two daughter plastids stochastically (
Ehara et al., 1990;
Kamimura et al., 2018). A similar morphological change of plastid nucleoids is also observed in the plant Arabidopsis (
Arabidopsis thaliana;
Terasawa and Sato, 2005), and thus, the mechanism of nucleoid segregation is apparently conserved in algae and plants. However, the molecular mechanisms underlying organelle DNA segregation and changes in nucleoid morphology have remained largely unknown.In a previous study using the green alga
C. reinhardtii, mutants defective in nucleoid segregation were isolated (
Misumi et al., 1999). One of the mutants possessed only a single enlarged nucleoid in a plastid (), which was inherited by daughter plastids unevenly (
Misumi et al., 1999). Later, the mutation responsible for this phenotype was identified in a previously uncharacterized gene,
Monokaryotic Chloroplast1 (
MOC1), which is conserved in eukaryotic algae and plants (
Kobayashi et al., 2017). MOC1 exhibited endonuclease activity in vitro, where it specifically cleaved Holliday junctions (HJs), four-stranded DNA structures formed during homologous recombination (HR;
Kobayashi et al., 2017). MOC1 symmetrically introduced nicks between consecutive cytosines (C↓C, where the arrow indicates the cleavage point) at the core of HJs (
Kobayashi et al., 2017). Because HJs provide a covalent link between recombining DNA molecules and must be removed prior to genome segregation (
Liu and West, 2004;
West, 2009), it was suggested that HR affects the nucleoid structure and MOC1 segregates plastid nucleoids by cleaving HJs between ptDNA molecules prior to plastid division (
Kobayashi et al., 2017).
Open in a separate windowMOC1 depletion destabilizes ptDNA by increasing ectopic recombination between SDRs in
C. reinhardtii. A, Differential interference contrast (DIC) and fluorescent images of SYBR Green I-stained wild-type (WT) and
MOC1 KO cells. Green is SYBR Green I-stained DNA and magenta is plastid chlorophyll fluorescence (Chl). The arrow indicates a plastic nucleoid. N, Nucleus. Scale bars = 5 μm. B, qPCR comparing relative copy number of ptDNA between the wild type and
CrMOC1 KO. The values of plastid
rpl2,
psbB,
chlN, and
psbD loci were normalized with that of the nuclear
CBLP locus. For
CrMOC1 KO, two independent clones (clones 1 and 2) were analyzed. The normalized value of the wild type was defined as 1.0. The error bar represents the mean ±
sd (
n = 3). Asterisks indicate significant difference by Student’s
t test (**
P < 0.01). C, Positions of SDRs CD5, CD5′, CI12, and CD15 in
C. reinhardtii ptDNA (
Odahara et al., 2016). Large inverted repeats (IRa and IRb) are shown by bold lines. SDRs are indicated by triangles. D to F, qPCR comparing relative copy numbers of ectopic recombinants of CD5 (D), CI12 (E), and CD15 (F). The recombinants were quantified with primers designed as shown in
Supplemental Figure S2. Each value was normalized with that of the plastid
psbB locus. The error bar represents the mean ±
sd (
n = 3). Asterisks indicate significant difference by Student’s
t test (ns,
P ≥ 0.05, *
P < 0.05, and **
P < 0.01).In general, HR follows either of two main pathways, the double-strand break repair (DSBR) or the synthesis-dependent strand annealing (SDSA) pathway (
Supplemental Fig. S1;
Holliday, 1964;
Szostak et al., 1983;
Pâques and Haber, 1999). The two pathways are similar in the initial step. After a double-strand break occurs, 5′ ends of the break are cut back to create 3′ overhangs of single-strand DNA (ssDNA). Recombinases bind the 3′ overhangs of ssDNA and search through vast quantities of DNA sequence to align and pair ssDNA with a homologous double-strand DNA template, facilitating the formation of a
d-loop (
Dunderdale et al., 1991;
Murayama et al., 2008). In the DSBR pathway, the end of the invading 3′ strand is extended by a DNA polymerase and converted into a HJ. The other 3′ overhang strand also forms a HJ with the homologous DNA. After that, there are two pathways to convert the double HJs into recombination products (
Sung and Klein, 2006). One pathway is mediated by HJ resolvases, which cleave HJs and produce either crossover or noncrossover products (
Szostak et al., 1983). Various HJ resolvases have been found in archaea, bacteria, and eukaryotes (
West, 2009). The eukaryotic nucleus also possesses another pathway, which is driven by the BTR complex consisting of the Bloom syndrome helicase, topoisomerase IIIα (TOP3A), and recombination-deficient Q-mediated genome instability subcomplex proteins (
Wu and Hickson, 2003). The BTR complex does not cleave but dissolves the double HJs (
Wu and Hickson, 2003). During the dissolution, the two HJ branches migrate toward each other until they form a hemicatenated intermediate, which is decatenated by TOP3A. Therefore, the dissolution pathway never produces crossover products (
Supplemental Fig. S1). In contrast to DSBR, in the SDSA pathway, the invading 3′ ssDNA is utilized as a primer and extended along the recipient DNA duplex by a DNA polymerase without forming HJs. The newly synthesized strand dissociates from the template DNA and anneals with the other 3′ overhang strand through complementary base pairing. After the strands anneal, the remaining single-stranded gaps are filled by a DNA polymerase (
Supplemental Fig. S1;
Pâques and Haber, 1999).In nuclear, mitochondrial, and plastid genomes, numerous short dispersed repeats (SDRs) exist (
Ottaviani et al., 2014). Thanks to the accuracy of recombinases in finding the homologous sequences despite the existence of myriad SDRs, the genomic sequence of the broken DNA is restored precisely via HR (
McEntee et al., 1979;
Shibata et al., 1979;
Qi et al., 2015). However, in the absence of recombinases, SDRs anneal through complementary base pairing and produce recombinants between SDRs (
Supplemental Fig. S1). Intramolecular recombination between SDRs results in deletion or inversion of the flanking region when they are oriented as direct or inverted repeats, respectively (
Supplemental Fig. S2). This pathway is known as microhomology-mediated end joining (MMEJ;
Supplemental Fig. S1;
McVey and Lee, 2008).Recombinase genes, including
Rad51 (eukaryotic type),
radA (archaeal type), and
recA (bacterial type), are believed to have evolved from a common ancestral gene (
Lin et al., 2006). Two phylogenetically distinct RECA proteins are encoded in the nuclear genome of land plants, of which one is targeted to plastids (ptRECA) and the other to mitochondria (mtRECA;
Lin et al., 2006). ptRECA and mtRECA are most closely related to cyanobacterial and proteobacterial counterparts, respectively, suggesting endosymbiotic origins of these proteins (
Lin et al., 2006). Two observations should be made regarding the functions of these two plant recombinases: (1) Suppression or loss of function of
ptRECA causes ectopic recombination between SDRs at a high frequency and destabilizes the plastid genome in the green alga
C. reinhardtii and the moss
Physcomitrella patens (
Odahara et al., 2015a,
2016), suggesting that ptRECA maintains integrity of the plastid genome by promoting HR and thus suppressing MMEJ. Because MOC1 possesses HJ-resolving activity in vitro and is required for segregation of ptDNA in vivo (
Kobayashi et al., 2017), RECA-mediated HR is likely accomplished with MOC1 through the DSBR pathway in plastids. However, how MOC1 functions in vivo has not been investigated. (2) Like ptRECA, mtRECA suppresses ectopic recombination between SDRs in the mitochondrial genome in
P. patens and the seed plant Arabidopsis (
Shedge et al., 2007;
Odahara et al., 2009;
Miller-Messmer et al., 2012). These results suggest that HJs are formed in plant mitochondria. However, except for CCE1, which is specific to yeasts (
Saccharomyces cerevisiae and
Schizosaccharomyces pombe;
Kleff et al., 1992;
Oram et al., 1998), mitochondrial HJ resolvases have not been identified in eukaryotes. Thus, it remains unclear whether HJs are formed and, if they are formed, how they are removed in mitochondria in plants.Regarding issues 1 and 2 described above, in this study, we show first that MOC1 suppresses ectopic recombination between SDRs in
C. reinhardtii plastids, as does ptRECA, suggesting that MOC1 is involved in DSBR in the plastid. Next, we show that MOC1 dually targets plastids and mitochondria in the moss
P. patens and maintains the integrity of ptDNA and mtDNA via suppression of ectopic recombination in both of these organelles. Putative dual-targeted transit peptides are also predicted in MOC1s of charophyte algae, a liverwort, a fern, and some seed plants, and we show that some of them are targeted to both plastids and mitochondria. Thus, it is suggested that HJs are formed during HR and removed by MOC1 in both plastids and mitochondria in some algae and plants.
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