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RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

Odahara M, Masuda Y, Sato M, Wakazaki M, Harada C, Toyooka K, Sekine Y - PLoS Genet. (2015)

Bottom Line: This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants.Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci.These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, College of Science, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo, Japan.

ABSTRACT
Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO) mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA) instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp) in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

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mtDNA rearrangements in RECG KO lines.A. mtDNA configuration at nad2, atp9 and ccmF loci. DNA from WT, RECG KO and RECA1 KO strains digested with EcoRI was probed using nad2, atp9 and ccmF probes, as indicated below the blots. The predicted structure and length of the major bands are indicated on the right. B. Schematic representation of the DNA structures detected in (A). The EcoRI fragments corresponding to the bands on the blot in (A) and the flanking regions are represented by solid black lines and dashed black lines, respectively. The EcoRI recognition sites are indicated by E. The positions of the probes used in (A) are indicated by thick gray lines. The boxes represent exons, and the lines between boxes represent introns or noncoding flanking sequences. The 69 bp and 47 bp repeats are indicated by black triangles and white triangles in the boxes, respectively. C. mtDNA configuration at the nad7 locus containing 79 bp repeats. DNA from each of the indicated strains was digested with SacII and probed using nad7 probe. The structure of the fragments is detailed in Odahara et al. [6]. D. Production of deleted mitochondrial subgenome by recombination between repeats in nad7 and nad9. Left panel illustrates production of deleted subgenome by intramolecular recombination between direct repeats. Undigested DNA from WT and RECG KO strains was probed using nad7 probe. The asterisk denotes DNA corresponding to 11-kb subgenome. c.z., compression zone. E. The amount of DNA generated by recombination between mtIR (62 bp). Relative copy number of DNA resulting from recombination between mtIR per mitochondrial rpl2 DNA was measured by qPCR. WT was given a value of 1. The data represent mean of three replicates ± SD. *p<0.01 (versus WT).
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pgen.1005080.g005: mtDNA rearrangements in RECG KO lines.A. mtDNA configuration at nad2, atp9 and ccmF loci. DNA from WT, RECG KO and RECA1 KO strains digested with EcoRI was probed using nad2, atp9 and ccmF probes, as indicated below the blots. The predicted structure and length of the major bands are indicated on the right. B. Schematic representation of the DNA structures detected in (A). The EcoRI fragments corresponding to the bands on the blot in (A) and the flanking regions are represented by solid black lines and dashed black lines, respectively. The EcoRI recognition sites are indicated by E. The positions of the probes used in (A) are indicated by thick gray lines. The boxes represent exons, and the lines between boxes represent introns or noncoding flanking sequences. The 69 bp and 47 bp repeats are indicated by black triangles and white triangles in the boxes, respectively. C. mtDNA configuration at the nad7 locus containing 79 bp repeats. DNA from each of the indicated strains was digested with SacII and probed using nad7 probe. The structure of the fragments is detailed in Odahara et al. [6]. D. Production of deleted mitochondrial subgenome by recombination between repeats in nad7 and nad9. Left panel illustrates production of deleted subgenome by intramolecular recombination between direct repeats. Undigested DNA from WT and RECG KO strains was probed using nad7 probe. The asterisk denotes DNA corresponding to 11-kb subgenome. c.z., compression zone. E. The amount of DNA generated by recombination between mtIR (62 bp). Relative copy number of DNA resulting from recombination between mtIR per mitochondrial rpl2 DNA was measured by qPCR. WT was given a value of 1. The data represent mean of three replicates ± SD. *p<0.01 (versus WT).

Mentions: A product resulting from recombination between 69 bp direct repeats existing in nad2 and atp9 loci of mtDNA, which appears as a 1.8 kb EcoRI fragment on DNA gel blots [6], was confirmed in a blot hybridized with nad2 probe in RECA1 KO lines (Fig. 5A). The blot showed that the 1.8 kb EcoRI fragment of the nad2-atp9 recombination product was hardly detectable in the RECG KO lines, while a weak 1.9 kb band was detected in both RECG KO lines (Fig. 5A). Since the RECG KO-specific 1.9 kb DNA fragment strongly hybridized to both an atp9 probe, which does not hybridize to the nad2-atp9 recombination product, and a ccmF probe (Fig. 5A), this fragment is likely to be the result of recombination between the atp9 locus and ccmF locus. Forty-seven base pair repeats at both loci are suspected to be involved in recombination (Fig. 5B and S4D Fig.), and the size of the recombination product is consistent with the size of the observed 1.9 kb ccmF-atp9 product. Because the ccmF-atp9 recombination product includes the 69 bp nad2-atp9 repeats (Fig. 5B), it hybridized weakly to the nad2 probe (Fig. 5A). The ccmF-atp9 product also appeared in the RECG KO lines with the sizes of the DNA products resulting from recombination between the 47 bp repeats when the mtDNAs were digested with HindIII or NdeI (S4A–C Fig.). PCR amplification of the ccmF-atp9 product from RECG KO plants and direct sequencing analysis of the amplified fragments showed that almost all of the recombination junctions were within the 47 bp repeats, yet the sequence similarity extended to the region flanking the repeat (S4D Fig.). These results indicate that the ccmF-atp9 recombination product, but not the nad2-atp9 recombination product, accumulates in the RECG KO lines.


RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

Odahara M, Masuda Y, Sato M, Wakazaki M, Harada C, Toyooka K, Sekine Y - PLoS Genet. (2015)

mtDNA rearrangements in RECG KO lines.A. mtDNA configuration at nad2, atp9 and ccmF loci. DNA from WT, RECG KO and RECA1 KO strains digested with EcoRI was probed using nad2, atp9 and ccmF probes, as indicated below the blots. The predicted structure and length of the major bands are indicated on the right. B. Schematic representation of the DNA structures detected in (A). The EcoRI fragments corresponding to the bands on the blot in (A) and the flanking regions are represented by solid black lines and dashed black lines, respectively. The EcoRI recognition sites are indicated by E. The positions of the probes used in (A) are indicated by thick gray lines. The boxes represent exons, and the lines between boxes represent introns or noncoding flanking sequences. The 69 bp and 47 bp repeats are indicated by black triangles and white triangles in the boxes, respectively. C. mtDNA configuration at the nad7 locus containing 79 bp repeats. DNA from each of the indicated strains was digested with SacII and probed using nad7 probe. The structure of the fragments is detailed in Odahara et al. [6]. D. Production of deleted mitochondrial subgenome by recombination between repeats in nad7 and nad9. Left panel illustrates production of deleted subgenome by intramolecular recombination between direct repeats. Undigested DNA from WT and RECG KO strains was probed using nad7 probe. The asterisk denotes DNA corresponding to 11-kb subgenome. c.z., compression zone. E. The amount of DNA generated by recombination between mtIR (62 bp). Relative copy number of DNA resulting from recombination between mtIR per mitochondrial rpl2 DNA was measured by qPCR. WT was given a value of 1. The data represent mean of three replicates ± SD. *p<0.01 (versus WT).
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pgen.1005080.g005: mtDNA rearrangements in RECG KO lines.A. mtDNA configuration at nad2, atp9 and ccmF loci. DNA from WT, RECG KO and RECA1 KO strains digested with EcoRI was probed using nad2, atp9 and ccmF probes, as indicated below the blots. The predicted structure and length of the major bands are indicated on the right. B. Schematic representation of the DNA structures detected in (A). The EcoRI fragments corresponding to the bands on the blot in (A) and the flanking regions are represented by solid black lines and dashed black lines, respectively. The EcoRI recognition sites are indicated by E. The positions of the probes used in (A) are indicated by thick gray lines. The boxes represent exons, and the lines between boxes represent introns or noncoding flanking sequences. The 69 bp and 47 bp repeats are indicated by black triangles and white triangles in the boxes, respectively. C. mtDNA configuration at the nad7 locus containing 79 bp repeats. DNA from each of the indicated strains was digested with SacII and probed using nad7 probe. The structure of the fragments is detailed in Odahara et al. [6]. D. Production of deleted mitochondrial subgenome by recombination between repeats in nad7 and nad9. Left panel illustrates production of deleted subgenome by intramolecular recombination between direct repeats. Undigested DNA from WT and RECG KO strains was probed using nad7 probe. The asterisk denotes DNA corresponding to 11-kb subgenome. c.z., compression zone. E. The amount of DNA generated by recombination between mtIR (62 bp). Relative copy number of DNA resulting from recombination between mtIR per mitochondrial rpl2 DNA was measured by qPCR. WT was given a value of 1. The data represent mean of three replicates ± SD. *p<0.01 (versus WT).
Mentions: A product resulting from recombination between 69 bp direct repeats existing in nad2 and atp9 loci of mtDNA, which appears as a 1.8 kb EcoRI fragment on DNA gel blots [6], was confirmed in a blot hybridized with nad2 probe in RECA1 KO lines (Fig. 5A). The blot showed that the 1.8 kb EcoRI fragment of the nad2-atp9 recombination product was hardly detectable in the RECG KO lines, while a weak 1.9 kb band was detected in both RECG KO lines (Fig. 5A). Since the RECG KO-specific 1.9 kb DNA fragment strongly hybridized to both an atp9 probe, which does not hybridize to the nad2-atp9 recombination product, and a ccmF probe (Fig. 5A), this fragment is likely to be the result of recombination between the atp9 locus and ccmF locus. Forty-seven base pair repeats at both loci are suspected to be involved in recombination (Fig. 5B and S4D Fig.), and the size of the recombination product is consistent with the size of the observed 1.9 kb ccmF-atp9 product. Because the ccmF-atp9 recombination product includes the 69 bp nad2-atp9 repeats (Fig. 5B), it hybridized weakly to the nad2 probe (Fig. 5A). The ccmF-atp9 product also appeared in the RECG KO lines with the sizes of the DNA products resulting from recombination between the 47 bp repeats when the mtDNAs were digested with HindIII or NdeI (S4A–C Fig.). PCR amplification of the ccmF-atp9 product from RECG KO plants and direct sequencing analysis of the amplified fragments showed that almost all of the recombination junctions were within the 47 bp repeats, yet the sequence similarity extended to the region flanking the repeat (S4D Fig.). These results indicate that the ccmF-atp9 recombination product, but not the nad2-atp9 recombination product, accumulates in the RECG KO lines.

Bottom Line: This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants.Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci.These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, College of Science, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo, Japan.

ABSTRACT
Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO) mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA) instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp) in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

Show MeSH
Related in: MedlinePlus