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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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Genomic instability in RECG KO plastids.A. ptDNA configuration at the ptIR-1 (rpl16) locus. DNA from WT and RECG KO strains digested with XhoI were probed with rpl16 probe. The asterisk denotes signals corresponding to DNA recombined between ptIR-1 repeats. The length of the bands is indicated on the left. B. Schematic explanation of the DNA structures detected in (A). Intramolecular recombination between ptIR-1 causes inversion of a segment between ptIR-1. The XhoI recognition sites are indicated by X. The boxes represent exons, and the lines between boxes represent introns or noncoding flanking sequences. ptIR-1 are indicated by black triangles in the boxes. The positions of the probes used in (A) are indicated by thick gray line. C. The amount of DNA generated by recombination between ptIR-1 (63 bp) or ptDR-1 (48 bp). Relative copy number of DNA per plastid ndhH 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). D. DNA generated by recombination between short (<35 bp) repeats. PCR reaction numbers indicated on the left of the pictures correspond to those in S3 Table. Plastid gene ndhH and nuclear gene actin were amplified as a control. Filled and blank triangles indicate DNA with the expected and unexpected sizes, respectively.
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pgen.1005080.g008: Genomic instability in RECG KO plastids.A. ptDNA configuration at the ptIR-1 (rpl16) locus. DNA from WT and RECG KO strains digested with XhoI were probed with rpl16 probe. The asterisk denotes signals corresponding to DNA recombined between ptIR-1 repeats. The length of the bands is indicated on the left. B. Schematic explanation of the DNA structures detected in (A). Intramolecular recombination between ptIR-1 causes inversion of a segment between ptIR-1. The XhoI recognition sites are indicated by X. The boxes represent exons, and the lines between boxes represent introns or noncoding flanking sequences. ptIR-1 are indicated by black triangles in the boxes. The positions of the probes used in (A) are indicated by thick gray line. C. The amount of DNA generated by recombination between ptIR-1 (63 bp) or ptDR-1 (48 bp). Relative copy number of DNA per plastid ndhH 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). D. DNA generated by recombination between short (<35 bp) repeats. PCR reaction numbers indicated on the left of the pictures correspond to those in S3 Table. Plastid gene ndhH and nuclear gene actin were amplified as a control. Filled and blank triangles indicate DNA with the expected and unexpected sizes, respectively.

Mentions: The fact that RECG protein not only localizes to mitochondria, but also to plastids, raises the possibility that RECG plays a role in both plastids and mitochondria. We then analyzed the structure of RECG KO ptDNA, and focused on recombination between repeated sequences. We searched for repeats using REPuter and identified 16 pairs of repeats longer than 40 bp in the P. patens ptDNA sequence [31], most of which were located immediately downstream of genes as palindromic sequences (S2 Table), probably functioning in the stabilization of transcripts [32]. Among the repeats, we analyzed the level of recombination between inverted repeats-1 (ptIR-1, 63 bp long, shown as R6 in S2 Table), which are located in rpl16 and trnG, or direct repeats-1 (ptDR-1, 48 bp long, shown as R12 in S2 Table), which are located in psaA and psaB (S6A Fig.). DNA gel blot analysis using a plastid rpl16 probe showed accumulation of 4.3 kb DNA fragments in the RECG KO mutants (Fig. 8A). The size of these DNA fragments corresponds to that of a predicted product resulting from recombination between ptIR-1 (Fig. 8B). We next analyzed the IR-1 recombination product using qPCR (S6 Fig.). The analyses revealed that the product formed by recombination between ptIR-1 showed ∼160-fold increase in the RECG KO mutants (Fig. 8C). Similar qPCR analysis of a product formed by recombination between ptDR-1 showed a 6–16-fold increase in the RECG KO mutants compared with WT (Fig. 8C). These results showed increased accumulation of ptIR-1 and ptDR-1 recombination products in the RECG KO lines. To assess the effect of RECG KO on copy number of ptDNA, qPCR analysis of ptDNA loci was performed. The copy number of three plastidic loci rbcL, atpA and ndhH showed increases in the RECG KO lines compared with WT (S6B Fig.). As the RECG KO mutants showed increase of ptDNA in every tested locus, it is possible that plastid number was increased in the RECG KO mutants. However, no significant difference was observed in the number of plastids per cell between WT and RECG KO mutants (S6C Fig.).


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)

Genomic instability in RECG KO plastids.A. ptDNA configuration at the ptIR-1 (rpl16) locus. DNA from WT and RECG KO strains digested with XhoI were probed with rpl16 probe. The asterisk denotes signals corresponding to DNA recombined between ptIR-1 repeats. The length of the bands is indicated on the left. B. Schematic explanation of the DNA structures detected in (A). Intramolecular recombination between ptIR-1 causes inversion of a segment between ptIR-1. The XhoI recognition sites are indicated by X. The boxes represent exons, and the lines between boxes represent introns or noncoding flanking sequences. ptIR-1 are indicated by black triangles in the boxes. The positions of the probes used in (A) are indicated by thick gray line. C. The amount of DNA generated by recombination between ptIR-1 (63 bp) or ptDR-1 (48 bp). Relative copy number of DNA per plastid ndhH 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). D. DNA generated by recombination between short (<35 bp) repeats. PCR reaction numbers indicated on the left of the pictures correspond to those in S3 Table. Plastid gene ndhH and nuclear gene actin were amplified as a control. Filled and blank triangles indicate DNA with the expected and unexpected sizes, respectively.
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Related In: Results  -  Collection

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Show All Figures
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pgen.1005080.g008: Genomic instability in RECG KO plastids.A. ptDNA configuration at the ptIR-1 (rpl16) locus. DNA from WT and RECG KO strains digested with XhoI were probed with rpl16 probe. The asterisk denotes signals corresponding to DNA recombined between ptIR-1 repeats. The length of the bands is indicated on the left. B. Schematic explanation of the DNA structures detected in (A). Intramolecular recombination between ptIR-1 causes inversion of a segment between ptIR-1. The XhoI recognition sites are indicated by X. The boxes represent exons, and the lines between boxes represent introns or noncoding flanking sequences. ptIR-1 are indicated by black triangles in the boxes. The positions of the probes used in (A) are indicated by thick gray line. C. The amount of DNA generated by recombination between ptIR-1 (63 bp) or ptDR-1 (48 bp). Relative copy number of DNA per plastid ndhH 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). D. DNA generated by recombination between short (<35 bp) repeats. PCR reaction numbers indicated on the left of the pictures correspond to those in S3 Table. Plastid gene ndhH and nuclear gene actin were amplified as a control. Filled and blank triangles indicate DNA with the expected and unexpected sizes, respectively.
Mentions: The fact that RECG protein not only localizes to mitochondria, but also to plastids, raises the possibility that RECG plays a role in both plastids and mitochondria. We then analyzed the structure of RECG KO ptDNA, and focused on recombination between repeated sequences. We searched for repeats using REPuter and identified 16 pairs of repeats longer than 40 bp in the P. patens ptDNA sequence [31], most of which were located immediately downstream of genes as palindromic sequences (S2 Table), probably functioning in the stabilization of transcripts [32]. Among the repeats, we analyzed the level of recombination between inverted repeats-1 (ptIR-1, 63 bp long, shown as R6 in S2 Table), which are located in rpl16 and trnG, or direct repeats-1 (ptDR-1, 48 bp long, shown as R12 in S2 Table), which are located in psaA and psaB (S6A Fig.). DNA gel blot analysis using a plastid rpl16 probe showed accumulation of 4.3 kb DNA fragments in the RECG KO mutants (Fig. 8A). The size of these DNA fragments corresponds to that of a predicted product resulting from recombination between ptIR-1 (Fig. 8B). We next analyzed the IR-1 recombination product using qPCR (S6 Fig.). The analyses revealed that the product formed by recombination between ptIR-1 showed ∼160-fold increase in the RECG KO mutants (Fig. 8C). Similar qPCR analysis of a product formed by recombination between ptDR-1 showed a 6–16-fold increase in the RECG KO mutants compared with WT (Fig. 8C). These results showed increased accumulation of ptIR-1 and ptDR-1 recombination products in the RECG KO lines. To assess the effect of RECG KO on copy number of ptDNA, qPCR analysis of ptDNA loci was performed. The copy number of three plastidic loci rbcL, atpA and ndhH showed increases in the RECG KO lines compared with WT (S6B Fig.). As the RECG KO mutants showed increase of ptDNA in every tested locus, it is possible that plastid number was increased in the RECG KO mutants. However, no significant difference was observed in the number of plastids per cell between WT and RECG KO mutants (S6C Fig.).

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