Expression and Complementation Analyses of a Chloroplast-Localized

Homolog of Bacterial RecA in the Moss Physcomitrella patens

Takayuki INOUYE,1 Masaki ODAHARA,1 Tomomichi FUJITA,2;*

Mitsuyasu HASEBE,2;3 and Yasuhiko SEKINE1;y

1Department of Life Science, College of Science, Rikkyo (St. Paul’s) University,

3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan2National Institute for Basic Biology, Okazaki 444-8585, Japan3Department of Basic Biology, The Graduate University for Advanced Studies,

Okazaki 444-8585, Japan

Received January 9, 2008; Accepted January 24, 2008; Online Publication, May 7, 2008

[doi:10.1271/bbb.80014]

RecA protein is widespread in bacteria, and it plays

a crucial role in homologous recombination. We

have identified two bacterial-type recA gene homologs

(PprecA1, PprecA2) in the cDNA library of the moss

Physcomitrella patens. N-terminal fusion of the putative

organellar targeting sequence of PpRecA2 to the

green fluorescent protein (GFP) caused a targeting of

PpRecA2 to the chloroplasts. Mutational analysis

showed that the first AUG codon acts as initiation

codon. Fusion of the full-length PpRecA2 to GFP caused

the formation of foci that were colocalized with

chloroplast nucleoids. The amounts of PprecA2 mRNA

and protein in the cells were increased by treatment

with DNA damaging agents. PprecA2 partially comple-

mented the recA mutation in Escherichia coli. These

results suggest the involvement of PpRecA2 in the

repair of chloroplast DNA.

Key words: Physcomitrella patens; homologous recom-

bination; DNA repair; chloroplast; RecA

Chloroplasts, which have evolved from endosymbio-sis of ancient cyanobacteria, have their own geneticsystems, replication, transcription, and translation, re-sembling those of bacteria. Although not extensivelystudied yet, it is reasonable that bacteria-type recombi-national repair systems function in chloroplasts. In fact,homologous recombination has been observed in achloroplast genome.1–3)

Homologous recombination is a process that occursin all organisms. It is responsible for genetic diversity,the maintenance of genome integrity, and the propersegregation of chromosomes.4) The functional relation-

ships between DNA replication and recombinationalprocesses has recently been recognized.5–8) RecA pro-tein, a crucial component in homologous recombinationand the related repair system in bacteria, binds to DNAto form a nucleoprotein filament that then aligns witha homologous duplex to promote single-strand ex-change.9,10) Recent study suggests that RecA and otherrecombination proteins also participate in the nonmuta-genic repair of stalled or collapsed replication forks.9)

Homologs of RecA have been identified in plantspecies,11) and functions of them in chloroplasts havebeen suggested. A plant Arabidopsis thaliana cDNAencoding for a bacterial RecA homolog, AtcpRecA(At1g79050), has been isolated,12) and was found to betargeted to chloroplasts.13) In pea chloroplasts, strandtransfer activity resembling that of bacterial RecA wasdetected14) and a protein cross-reacting with the Esche-richia coli RecA antibody was identified.15) A RecAdependent recombination system has also been suggest-ed for the chloroplast of Chlamydomonas reinhardtii,unicellular green algae,16) and the C. reinhardtii chloro-plast RecA was identified.17) Thus it is likely that anRecA-dependent recombination and repair system isubiquitous and well conserved among chloroplastgenetic systems, but little is known about the biologicalsignificance of homologous recombination in chloro-plasts.A moss, Physcomitrella patens, has become a model

plant in the study of gene function by targeted genedisruption, which occurs with efficiency similar tothat observed in yeast.18) We have reported that twoP. patens RecA homologs, PpRecA1 and PpRecA2,have been identified, and that PpRecA1 was specifically

* Present address: Graduate School of Science, Hokkaido University, Kita 10 Nishi 8, Sapporo 060-0810, Japan

y To whom correspondence should be addressed. Fax: +81-3-3985-3408; E-mail: ysekine@rikkyo.ac.jp

Abbreviations: cpDNA, chloroplast DNA; DAPI, 40, 6-diamidino-2-phenylindole; DSBs, double strand breaks; GFP, green fluorescent protein;

MMS, methyl methanesulfonate; RT-PCR, reverse transciptase-polymerase chain reaction; TBS, Tris-buffered saline

Biosci. Biotechnol. Biochem., 72 (5), 1340–1347, 2008

localized to mitochondria.19) Here we report the analysisof a PprecA2 cDNA encoding a putative chloroplastRecA protein. Analysis of the RecA in this model plantcells should open ways to understand the chloroplastDNA maintenance system in plant cells.

Materials and Methods

Plant materials, growth conditions, and DNA damagetreatments. Protonemata of Physcomitrella patens Bruchand Schimp subsp. patens were cultured on cellophane-overlaid BCDAT medium (1mM MgSO4, 10mM KNO3,45 mM FeSO4, 1.8mM KH2SO4, 1mM CaCl2, 5mM

ammonium tartrate, and 0.8% agar).20) All plants weregrown at 25 �C under constant light conditions (70mmol photonsm�2 s�1). For semi-quantitative RT-PCRor Western blot analysis of PprecA2, methyl methanesulfonate (MMS) or bleomycin treatments were per-formed, as described previously.19)

Construction of GFP-fusion plasmids. A DNA frag-ment containing the green fluorescent protein (GFP)gene, in which the initiation codon ATG is convertedto TTG, was obtained by KpnI-EcoRI digestion ofpGFPmutNPTII (T. Fujita, unpublished), and was thencloned into KpnI-EcoRI cleaved p7133-PprecA1-sGFP,19) a derivative of p7133TP-sGFP21) carrying thePprecA1 cDNA segment, yielding p7133-sGFP (TTG).A cloned cDNA corresponding to PprecA2, obtainedfrom the full-length cDNA library of P. patens,22) wasused as template for PCR amplification. The first 500 bpof PprecA2 cDNA, encompassing the coding regionfor the putative signal peptide, was amplified usingprimers 50-GGGGGTACCCTGTTTCTGACCTCGCT-GCTATG-30 (named PprecA2-F001) and 50-GGGATC-GATGCCATTGATGTCGCTCATCGC-30 (PprecA2-R04), in which the restriction sites are underlined.The PCR product was cloned into KpnI-ClaI cleavedp7133-sGFP (TTG), yielding pINO44.

Plasmids pINO46 (ATG) and pINO47, derivatives ofpINO44 carrying mutations converting the first andsecond ATG codons in the PprecA2 cDNA to TTGrespectively, were constructed as follows: Mutationswere introduced into the cloned PprecA2 cDNA with aQuikChange Site-Directed Mutagenesis Kit (Stratagene,La Jolla, CA) using primers 50-CTGCAATTGGC-TTGGCAGCAATAGC-30 and 50-GCTATTGCTGC-CAAGCCAATTGCAG-30 for mutagenesis of the firstATG, yielding pINO41, or using primers 50-CAACC-TGCGCTTGCCAAGCCCGTC-30 and 50-GACGGGC-TTGGCAAGCGCAGGTTG-30 for mutagenesis of thesecond ATG, yielding pINO42. Next, the first 500 bpsegment of the PprecA2 cDNA in the resulting plasmids,pINO41 and pINO42, was amplified using primersPprecA2-F001 and PprecA2-R04. The PCR productamplified from pINO41 was cloned into KpnI-ClaIcleaved p7133-PprecA1-sGFP,19) yielding pINO46(ATG). The PCR product amplified from pINO42 was

cloned into KpnI-ClaI cleaved p7133-sGFP (TTG),yielding pINO47.The full-length PprecA2 coding region was amplified

by PCR using cloned PprecA2 cDNA as template andprimers 50-CCCGGTACCGCCGGCCCGGGGGCAT-GGCAGCAATAGCTGTGC-30 and 50-CCCGAGCTC-CATGGCCCGGGAGTCAAGCTCTCCCAAATTGTC-30. The PCR product was cloned into KpnI-NcoI cleavedp7133-PprecA1-sGFP,19) yielding pMAK125.

Transient expression of GFP-fusion constructs inP. patens and fluorescent microscopy observation. Thegeneration of P. patens protoplasts and the subsequenttransformation were performed as described previous-ly.20) Two days after transformation, the GFP, chloro-phyll, and 40, 6-diamidino-2-phenylindole (DAPI) fluo-rescence of the protoplasts was observed using anOlympus AX80 microscope (Olympus, Tokyo). ForDAPI staining, protoplasts were fixed with 1% gluta-raldehyde (Wako, Osaka, Japan) dissolved in TANbuffer (20mM Tris–HCl, pH 7.65, 0.5mM EDTA, 7mM

2-mercaptoetanol, 0.4mM phenylmethylsulfonyl fluo-ride, and 1.2mM spermidine) and were stained with0.2 mg/ml of DAPI for 10min, followed by fluorescentmicroscopy observation.

Semi-quantitative reverse transciptase-PCR (RT-PCR) for expression analysis. Total RNA was isolatedfrom wild-type protonemata with the RNeasy PlantMini Kit (Qiagen, Valencia, CA). Reverse transcriptionand subsequent PCR were performed as describedpreviously.19) To amplify a segment of PprecA2,primers 50-CCCTCTAGACCGGTTAATGAGTCGA-GC-30 and 50-GAAATCCTACAACCACAGCAACC-ATTCAG-30 were used. PCR amplification was per-formed in the exponential amplification phase. The PCRproducts were analyzed by electrophoresis in an agarosegel, and the gel was stained with SYBR Green I (TakaraBio Inc., Otsu, Japan). The integrated intensities ofthe product bands were determined using Typhoon 9210and Image Quant (GE Healthcare, Little Chalfont, UK).

Antisera generation. For PCR amplification of thecoding segment of the PpRecA2 C-terminal region(amino acids 321-460), cloned PprecA2 cDNA astemplate and primers 50-CCCCCCATATGACGGG-CAAGATTAAGTCGCC-30 and 50-CCCCTGCAGT-GATCAGGAGCTCTGGTCAAGCTCTCCCAAATTG-TC-30 (PprecA2-30PBS) were used. The PCR productwas cloned into NdeI-SacI cleaved with pET-22b(Novagen, Inc., Madison, WI, USA) to produce C-terminal 6 � His-tagged recombinant protein, yieldingpINO21. The recombinant protein was purified with aNi-NTA agarose (Qiagen) according to the manufac-ture’s instructions. The protein was further purified bySDS–PAGE, and the excised protein in the gel slicewas used to immunize the rabbits.

RecA in Moss Chloroplasts 1341

Western blot analysis. Protonema were flash-frozen inliquid nitrogen and disrupted with a Multi-beads shocker(MB400U, Yasui-kikai, Osaka, Japan). Soluble plantproteins were extracted in 50mM Tris–HCl, pH 7.5,5mM EDTA, 10mM �-mercaptoethanol, and 1mM

phenylmethylsulfonyl fluoride. Samples were separatedinto supernatant and pellet fractions by centrifugation at20;000� g for 20min. All procedures were performedon ice or at 4 �C. Protein concentrations were measuredwith Coomassie plus protein assay reagent (PierceChemical, Rock-ford, IL, USA). For Western blotanalysis, 50 mg of soluble proteins was separated in12% polyacrylamide gels and electroblotted onto poly-vinylidene difluoride membrane (Millipore, Bedford,MA, USA). The membranes were blocked in 5% (w/v)skimmed milk powder in Tris-buffered saline (TBS;20mM Tris–HCl, 150mM NaCl, pH 7.5) for 1 h, andincubated with anti-PpRecA2 antiserum at a dilution of1:1,000 in TBS as primary antibody reaction, then ingoat anti-Rabbit antibody conjugated with alkalinephosphatase at a dilution of 1:5,000 in TBS. Washeswere performed with three changes of TBS. Thealkaline phosphatase reaction was visualized using theAttophos Fluorescent AP substrate system (Promega,Madison, WI, USA). The integrated intensities of thesignal were determined by Typhoon 9210 and ImageQuant.

Assay for complementation of E. coli recA deficientstrain. The PprecA2 gene was amplified by PCR withprimers 50-GGGCCATGGCAAAAGCCAAGAAAG-GA-30 and PprecA2-30PBS, and the PCR product wascloned between the NcoI and PstI sites of pBAD24,23)

yielding pINO1. E. coli CSR603 [recA1, uvrA6, phr-1]24) cells bearing pINO1, pMAK13219) carrying E. colirecA or pBAD24 were cultured to OD660 of 0.5 in M9medium25) containing 0.2% glucose and then transferredto M9 medium25) containing 0.2% arabinose andincubated for 30min at 37 �C for gene induction. Cellswere treated with different concentrations of mitomycinC and incubated for 30min at 37 �C. After washing,appropriate dilutions of treated cells were spread on LBplates and incubated overnight at 37 �C. Colony-formingunits were counted, and the surviving fraction of cellswas calculated as described previously.19)

Results

Identification of Physcomitrella patens recA (Pprec-A2)

By means of a BLAST search of full-length enrichedcDNA libraries of P. patens,22) we have identified twodistinct recA homologs which we named PprecA1 andPprecA2.19) The PprecA2 cDNA had two putativetranslation initiation sites in its 50 region and encodeda 460-residue or a 417-residue protein. Except for anN-terminal peptide, which was predicted to be achloroplast transit peptide, the putative gene product

shared high similarity with E. coli RecA protein andchloroplast RecA proteins from A. thaliana and C. rein-hardtii (Fig. 1A). We determined the genomic DNAsequence encompassing PprecA2 and compared it withthe cDNA sequence. The PprecA2 gene was composedof 13 exons and 12 introns, and the locations of theintrons corresponded well with those of AtcprecA(At1g79050) (Fig. 1B).

Subcellular localization of PpRecA2-GFPTo determine the subcellular localization of

PpRecA2, we prepared GFP fusion constructs contain-ing the 50-upstream sequence. The first 500 bp of thePprecA2 cDNA sequence, encompassing the codingregion for the putative signal peptide, was fused to theGFP gene under the control of a modified cauliflowermosaic virus 35S promoter, E7133.26) The construct wasintroduced into P. patens protoplasts by polyethyleneglycol-mediated transformation. The subcellular distri-butions of PpRecA2-GFP were completely included inthe areas of red chlorophyll fluorescence, indicating thatPpRecA2 was targeted to the chloroplasts (Fig. 2A, top).Using this construct, we did not detect the fluorescenceof the PpRecA2-GFP in the mitochondria.There exist two in-frame ATG codons in the 50

region of PprecA2. Both of the translated productswere predicted to be targeted to chloroplasts accordingto TargetP27) (the scores for the larger and smaller oneswere 0.957 and 0.620 respectively). The nucleotidesequence context of the AUG plays a role in theefficiency of translation initiation, but it was difficult toestimate the functional initiation codon by a comparisonof plant consensus sequences around AUG.28) Toexamine the translation initiation site, we preparedGFP fusion constructs in which one of the ATG codonswas mutated, and introduced it into P. patens proto-plasts. Using the construct with a mutation in the firstATG codon, faint green fluorescence was detected(Fig. 2A, middle). On the other hand, using the constructwith a mutation in the second ATG codon, strongfluorescence was detected in the chloroplasts (Fig. 2A,bottom), similarly, to the result of the construct withoutthe mutation. These results indicate that despite thepresence of two in-frame ATGs in the 50 region ofPprecA2, the first ATG is recognized as the initiationsite, resulting in the production of a protein that istargeted to chloroplasts.To examine further the subcellular localization of

PpRecA2 as a whole protein, we prepared a GFPconstruct containing full-length PprecA2, which encom-passes the entire coding region, and introduced itinto P. patens protoplasts. The green fluorescence ofPpRecA2-GFP formed small foci in chloroplasts(Fig. 2B). By staining with DAPI, the foci werecolocalized with chloroplast nucleoids (Fig. 2B). Thisresult indicates that PpRecA2 in the chloroplasts isassociated with chloroplast nucleoids.

1342 T. INOUYE et al.

Enhanced expression of PprecA2 after DNA damageExpression of the mitochondrial PprecA1 has been

shown to be enhanced after DNA damage.19) Moreover,the recA homologs of pea, A. thaliana, and C. rein-hardtii chloroplasts perhaps increase after DNA dam-age.12,13,17) To examine PprecA2 expression in DNA-damaged cells of P. patens, we analyzed the level ofPprecA2 expression in MMS- and bleomycin-treatedcells by semi-quantitative RT-PCR and immunoblot

analysis using anti-PpRecA2 antiserum. MMS, a DNAmethylating agent, perhaps causes double strand breaks(DSBs) by forming methylated bases in DNA,29) whilebleomycin, a radiomimetic DNA-damaging agent, caus-es DSBs directly.30) Total RNA and total solubleproteins were extracted from protonemata treated withMMS or bleomycin for 6 h, and semi-quantitative RT-PCR or immunoblot was performed. PprecA2 expres-sion was strongly induced by treatment with MMS and

A

B

Fig. 1. Bacterial RecA Homologs.

A, Alignment of the amino acid sequence of two RecA homologs from P. patens (PpRecA1, PpRecA2), chloroplast RecA homologs

from A. thaliana (AtcpRecA, At1g79050) and C. reinhardtii (CrRecA), and E. coli RecA. The accession numbers are PpRecA1 (AB284534),

PpRecA2 (AB284535), AtcpRecA (NP 001077844), CrRecA (AB048829), and E. coli RecA (AP 003266). Sequence identities are inverted.

The conserved functional domains of RecA9) are indicated under the sequences: Walker A and B are involved in ATP hydrolysis, and loop 1 and

loop 2 are important for DNA binding. The box represents the second methionine in the putative transit peptide of PpRecA2, which was

mutated to leucine as in Fig. 2. B, Gene organizations of PprecA2 and AtcprecA (At1g79050). The filled boxes and the lines between the boxes

represent exons and introns respectively. The corresponding exons are joined to each other by lines.

RecA in Moss Chloroplasts 1343

with bleomycin (Fig. 3A). Correspondingly, the amountof PpRecA2 protein in MMS- or bleomycin-treatedcells increased by up to about 3-fold as compared withnon-treated cells (Fig. 3B). These results suggest thatPprecA2 is involved in DNA repair.

Complementation of an E. coli recA deficient strainby PprecA2

To determine the functional similarities betweenchloroplast RecAs and their bacterial counterpart,complementation analysis was performed with anE. coli recA deficient strain. The cDNA fragmentencoding the PpRecA2 protein without the putativesignal peptide was placed under the control of thearabinose promoter, and the construct was transformedinto an E. coli recA deficient strain. Survival rates weremeasured after treating the cells with the DNA cross-linking agent mitomycin C. Upon the addition ofarabinose, the survival fraction of E. coli recA deficientcells bearing the PprecA2 plasmid increased by up to100-fold as compared to that of the strain bearing aplasmid lacking PprecA2, whereas no increase wasobserved when PprecA2 expression was repressed by

the addition of glucose (Fig. 4). These results suggestthat PpRecA2 partially complements the function ofE. coli RecA.

Discussion

In this study, we identified a Physcomitrella homologof the bacterial-type RecA gene. The gene productshares extensive sequence similarity with E. coli RecAprotein and chloroplast RecA from A. thaliana12) andC. reinhardtii17) except for the amino-terminal region.The PprecA2 transcript possesses two in-frame AUGcodons in the 50 terminal region. It has been reportedthat 50 sequences of phage-type RNA polymerase(PpRPOT1 and PpRPOT2) genes in P. patens alsocontained two in-frame AUG codons, and that both ofproteins were translated from the second AUG codonand targeted to mitochondria.31) It has also been reportedthat an A. thaliana RecA homolog, At2g19490, is dual-targeted to mitochondria and chloroplasts.32) In vivoexperiments with constructs containing the mutated firstAUG codon or the mutated second one revealed thatPpRecA2 was translated from the first ATG codon, and

A

B

Fig. 2. Subcellular Localization of PpRecA2-GFP in Transiently Transformed Protoplasts.

Structures of DNA constructs producing PpRecA2 (putative transit peptide)-GFP (A) and PpRecA2 (full-length)-GFP (B) are shown in the left

part of the figure. Open and gray rectangles represent the PprecA2 cDNA and sGFP genes, respectively. Maps are not drawn to scale, for

readability. The position of the A of the first ATG codon was defined as 1. Crosses over ATG indicate that the ATG codon was mutated to TTG.

P. patens protoplasts were transformed with the various PprecA2-GFP fusion constructs. Fluorescence of GFP, chlorophyll, and DAPI was

observed using a fluorescence microscope. Bar, 10mm.

1344 T. INOUYE et al.

that this protein was targeted only to chloroplasts (seeFig. 2A). Using these constructs, we did not detect thefluorescence of PpRecA2-GFP in the mitochondria. On

the other hand, fluorescence of PpRecA1-GFP wasdetected only in the mitochondria.19) On the basis ofthese lines of evidence, we concluded that PprecA2 geneencodes a bacterial-type RecA homolog that functions inthe chloroplasts. This is consistent with a phylogeneticanalysis suggesting that PpRecA2 is closely related tochloroplast RecA and cyanobacterial RecA.19)

We observed full-length PpRecA2-GFP focus forma-tion in P. patens chloroplasts (Fig. 2B). The foci werecolocalized with chloroplast nucleoids, suggesting thatPpRecA2 is bound to chloroplast DNA (cpDNA). InE. coli and Bacillus subtilis, RecA-GFP fusion proteinshave been visualized as foci in response to DNA damageand replication of damaged DNA.33,34) The PpRecA2-GFP foci may form in response to cpDNA damage,which might be caused in the course of the procedure forprotoplast preparation and subsequent transformation.As found in this study, the amount of PprecA2

transcript as well as that of the PpRecA2 protein wasincreased upon the addition of DNA-damaging agents(see Fig. 3), indicating that PprecA2 is induced by DNAdamage. Since DNA-damaging agents, such as MMSand bleomycin, induce lesions not only in cpDNA butalso in nuclear DNA, we cannot state with certainty thatthe lesions in cpDNA function as a signal for PprecA2induction. Furthermore, these agents generate reactiveoxygen species, which might be the inducer of PprecA2.Specific damage to cpDNA is required to elucidate theregulatory mechanism that responds to DNA damage.As presented in this study, PprecA2 partially com-

plemented an E. coli recA deficient strain, and this, toour knowledge, is the first time chloroplast RecA has

A

B

Fig. 3. Expression of PprecA2 in MMS- and Bleomycin-Treated Cells.

A, Total RNA was extracted from protonemata cultivated in the presence (+) or absence (�) of DNA damaging agents, MMS (20mM) or

bleomycin (25mg/ml). The GAPDH gene was amplified as an internal control. PCR was performed for 28 cycles for amplification of PprecA2

and for 22 cycles for the amplification of GAPDH. B, Western blot analysis of PpRecA2 in MMS- and bleomycin-treated cells. Equal amounts of

soluble proteins, which were extracted from protonemata cultivated in the presence (+) or absence (�) of MMS (20mM) or bleomycin (25mg/ml) were used.

Fig. 4. Partial Complementation of an E. coli recA Deficient Strain

by PprecA2.

The surviving fraction after mitomycin C treatment of the

CSR603 strain (recA�) harboring pMAK132 with E. coli recA

(square), pINO1 with PprecA2 (triangle), or pBAD24 (circle) alone

was calculated as described.19) The strain harboring pINO1 was

grown in a medium containing 0.2% arabinose (closed triangle) or

0.2% glucose (open triangle). The other strains were grown in a

medium containing 0.2% arabinose. Data are expressed as mean�S.D. (n ¼ 3).

RecA in Moss Chloroplasts 1345

been found to substitute for E. coli RecA in vivo. Thissuggests that PpRecA2 is a functional homolog ofE. coli RecA. Recombinational repair processes canalleviate blocks to DNA replication imposed by geno-toxic agents.5–9) Lesions in DNA, such as interstrandcross links produced by mitomycin C, can block DNApolymerases during replication, leading to the accumu-lation of single-strand DNA gaps or DSBs. It is possiblethat PpRecA2 does act in repair of stalled or collapsedreplication forks. The degree of complementation (up to100-fold) was high as compared to that (up to 10-fold) ofPpRecA1.19) This suggests that PpRecA2 can readilyinteract with E. coli recombinational factors such asRecBCD and/or RecFOR, since the amino acid se-quence similarity between PpRecA2 and E. coli RecAwas higher than that between PpRecA1 and E. coliRecA (Fig. 1A). However, the degree of complementa-tion was low as compared to that of E. coli RecA (seeFig. 4). E. coli RecA, which plays a central role inrecombinational repair pathways, is also involved in theSOS response, which also contributes to DNA repair.10)

This suggests that the greater survival rate of the E. colirecA deficient strain transformed with the E. coli recAexpression plasmid might be due to the SOS response.The presence of an SOS response in chloroplast isunclear, and cyanobacteria do not have an E. coli-typeSOS regulon,35) suggesting that PpRecA2 is unable toinduce the SOS response in E. coli.

Disruption of PprecA1 by targeted replacementresulted in lower rates of recovery of mitochondrialDNA from MMS damage, suggesting the involvementof PpRecA1 in the repair of mitochondrial DNA.19) Toelucidate a possible role of PpRecA2 in the maintenanceof cpDNA in P. patens, studies with targeting disrup-tants are currently in progress.

Acknowledgments

This work was supported by the Frontier Project‘‘Adaptation and Evolution of Extremophile’’ (to Y. S.),and by Grants-in-Aid for Creative Scientific Research(17GS0314 to Y.S.) from the Japan Society for thePromotion of Science.

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