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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 and RECA1 KO mitochondria.A. mtDNA configuration at the nad4 locus. DNA from WT, RECG KO, and RECA1 KO strains digested with SacII were probed with nad4 probe. The asterisks denote signals corresponding to DNA recombined between nad4-nad1 repeats. The length of the bands is indicated on the left. B. Schematic explanation of the DNA structures detected in (A). Reciprocal recombination at R4 (90 bp) or R6 (56 bp) at nad4 and nad1 loci produces both nad4-nad1 rec and nad1-nad4 rec products. The SacII recognition sites are indicated by S. The positions of the probes used in (A) are indicated by thick gray lines. For details of the scheme, see legend of Fig. 5B. C. The amount of DNA generated by recombination between several direct repeats 46 to 57 bp in length. Relative copy number of DNA resulting from recombination between direct repeats (R5, R11, R12, R13, R18, or R19) per mitochondrial rpl2 DNA was measured by qPCR. WT was given a value of 1. The data represent mean of three replicates ± SD. All the RECG KO and RECA1 KO values are significantly different from WT values (p<0.01). D. DNA generated by recombination between short (<35 bp) repeats. PCR reaction numbers indicated on the left correspond to those in S1 Table. Mitochondrial gene rpl2 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.g006: Genomic instability in RECG and RECA1 KO mitochondria.A. mtDNA configuration at the nad4 locus. DNA from WT, RECG KO, and RECA1 KO strains digested with SacII were probed with nad4 probe. The asterisks denote signals corresponding to DNA recombined between nad4-nad1 repeats. The length of the bands is indicated on the left. B. Schematic explanation of the DNA structures detected in (A). Reciprocal recombination at R4 (90 bp) or R6 (56 bp) at nad4 and nad1 loci produces both nad4-nad1 rec and nad1-nad4 rec products. The SacII recognition sites are indicated by S. The positions of the probes used in (A) are indicated by thick gray lines. For details of the scheme, see legend of Fig. 5B. C. The amount of DNA generated by recombination between several direct repeats 46 to 57 bp in length. Relative copy number of DNA resulting from recombination between direct repeats (R5, R11, R12, R13, R18, or R19) per mitochondrial rpl2 DNA was measured by qPCR. WT was given a value of 1. The data represent mean of three replicates ± SD. All the RECG KO and RECA1 KO values are significantly different from WT values (p<0.01). D. DNA generated by recombination between short (<35 bp) repeats. PCR reaction numbers indicated on the left correspond to those in S1 Table. Mitochondrial gene rpl2 and nuclear gene actin were amplified as a control. Filled and blank triangles indicate DNA with the expected and unexpected sizes, respectively.

Mentions: To analyze the effect of RECG or RECA1 KO on global structure of mtDNA, we performed a comprehensive analysis of DNA molecules resulting from recombination between repeats dispersed in the mtDNA. We first analyzed mtDNA repeat-mediated rearrangements in both KO mutants by DNA gel blot, and identified two DNA fragments that were most likely to be derived from recombination between nad4-nad1 direct repeats, named R4 (90 bp) or R6 (56 bp) [6], as judged by their sizes (Fig. 6A). Note that the DNA fragments were detected only in RECA1 KO lines, but not in RECG KO line as well as WT. We next carried out quantification of DNA resulting from recombination between other direct repeats (46–57 bp, R5, R11, R12, R13, R18, and R19) by qPCR. The results showed that recombined DNA from every tested repeats were apparently accumulated in both RECG and RECA1 KO lines; the level of accumulation was very high in the RECA1 KO lines regarding R5 and R13, and high in RECA1 KO lines regarding R11 and R18 (Fig. 6C). Collectively, these results suggest that the repeat-mediated recombination were induced in both RECG and RECA1 KO mitochondria at multiple loci, but the degree and the site of the recombination were somewhat different between them.


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 and RECA1 KO mitochondria.A. mtDNA configuration at the nad4 locus. DNA from WT, RECG KO, and RECA1 KO strains digested with SacII were probed with nad4 probe. The asterisks denote signals corresponding to DNA recombined between nad4-nad1 repeats. The length of the bands is indicated on the left. B. Schematic explanation of the DNA structures detected in (A). Reciprocal recombination at R4 (90 bp) or R6 (56 bp) at nad4 and nad1 loci produces both nad4-nad1 rec and nad1-nad4 rec products. The SacII recognition sites are indicated by S. The positions of the probes used in (A) are indicated by thick gray lines. For details of the scheme, see legend of Fig. 5B. C. The amount of DNA generated by recombination between several direct repeats 46 to 57 bp in length. Relative copy number of DNA resulting from recombination between direct repeats (R5, R11, R12, R13, R18, or R19) per mitochondrial rpl2 DNA was measured by qPCR. WT was given a value of 1. The data represent mean of three replicates ± SD. All the RECG KO and RECA1 KO values are significantly different from WT values (p<0.01). D. DNA generated by recombination between short (<35 bp) repeats. PCR reaction numbers indicated on the left correspond to those in S1 Table. Mitochondrial gene rpl2 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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pgen.1005080.g006: Genomic instability in RECG and RECA1 KO mitochondria.A. mtDNA configuration at the nad4 locus. DNA from WT, RECG KO, and RECA1 KO strains digested with SacII were probed with nad4 probe. The asterisks denote signals corresponding to DNA recombined between nad4-nad1 repeats. The length of the bands is indicated on the left. B. Schematic explanation of the DNA structures detected in (A). Reciprocal recombination at R4 (90 bp) or R6 (56 bp) at nad4 and nad1 loci produces both nad4-nad1 rec and nad1-nad4 rec products. The SacII recognition sites are indicated by S. The positions of the probes used in (A) are indicated by thick gray lines. For details of the scheme, see legend of Fig. 5B. C. The amount of DNA generated by recombination between several direct repeats 46 to 57 bp in length. Relative copy number of DNA resulting from recombination between direct repeats (R5, R11, R12, R13, R18, or R19) per mitochondrial rpl2 DNA was measured by qPCR. WT was given a value of 1. The data represent mean of three replicates ± SD. All the RECG KO and RECA1 KO values are significantly different from WT values (p<0.01). D. DNA generated by recombination between short (<35 bp) repeats. PCR reaction numbers indicated on the left correspond to those in S1 Table. Mitochondrial gene rpl2 and nuclear gene actin were amplified as a control. Filled and blank triangles indicate DNA with the expected and unexpected sizes, respectively.
Mentions: To analyze the effect of RECG or RECA1 KO on global structure of mtDNA, we performed a comprehensive analysis of DNA molecules resulting from recombination between repeats dispersed in the mtDNA. We first analyzed mtDNA repeat-mediated rearrangements in both KO mutants by DNA gel blot, and identified two DNA fragments that were most likely to be derived from recombination between nad4-nad1 direct repeats, named R4 (90 bp) or R6 (56 bp) [6], as judged by their sizes (Fig. 6A). Note that the DNA fragments were detected only in RECA1 KO lines, but not in RECG KO line as well as WT. We next carried out quantification of DNA resulting from recombination between other direct repeats (46–57 bp, R5, R11, R12, R13, R18, and R19) by qPCR. The results showed that recombined DNA from every tested repeats were apparently accumulated in both RECG and RECA1 KO lines; the level of accumulation was very high in the RECA1 KO lines regarding R5 and R13, and high in RECA1 KO lines regarding R11 and R18 (Fig. 6C). Collectively, these results suggest that the repeat-mediated recombination were induced in both RECG and RECA1 KO mitochondria at multiple loci, but the degree and the site of the recombination were somewhat different between them.

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