of Pea (Pisum sativum 1.) Protoplasts with DNA- Damaging ...Plant Physiol. (1993) 102: 155-163 Treatment of Pea (Pisum sativum 1.)Protoplasts with DNA- Damaging Agents lnduces a 39-Kilodalton

Plant Physiol. (1993) 102: 155-163

Treatment of Pea (Pisum sativum 1.) Protoplasts with DNA- Damaging Agents lnduces a 39-Kilodalton Chloroplast

Protein lmmunologically Related to Escherichia coli RecA'

Heriberto Cerutti', Hesham Z. Ibrahim3, and Andre T. Jagendorf*

Section of Plant Biology, Cornell University, Ithaca, New York 14853

Organisms must have efficient mechanisms of DNA repair and recombination to prevent alterations in their genetic information due to DNA damage. There is evidence for DNA repair and recombination in plastids of higher plants, although very little is known at the biochemical level. Many chloroplast proteins are of eubacterial ancestry, suggesting that the same could be true for the components of a DNA repair and recombination system. A 39- kD protein, immunologically related to Escherichia coli RecA, is present in chloroplasts of pea (Pisum safivum L.). Bandshift gel assays suggest that it binds single-stranded DNA. Its steady-state level is increased by several DNA-damaging agents. These results are consistent with it being a plastid homolog of E. coli RecA protein, presumably involved in DNA repair and recombination, and with the existence of an SOS-like response in pea leaf cells. Experiments with protein synthesis inhibitors suggest that the 39- kD chloroplast protein is encoded in the nucleus.

The DNA of a cell undergoes significant damage solely as a result of thermal fluctuations and normal metabolic activity (Lindahl and Nyberg, 1972; McLennan, 1988; Richter et al., 1988). Moreover, many chemical or physical agents can cause DNA damage (Saffhill et al., 1985; McLennan, 1988). To maintain accurately their genetic information, plants must have efficient mechanisms of DNA repair presumably oper- ating in each of the different DNA-containing organelles. Eubacteria, yeast, and mammalian cells possess several pathways of DNA repair, namely, direct repair (like photoreacti-

' Partia1 support was provided by Hatch grant 0155928 and by grant 91-37301-6421 from the U.S. Department of Agriculture/ National Research Initiative Competitive Grants Program. H.C. was supported by a predoctoral fellowship from the Cornell National Science Foundation Plant Science Center, a unit in the U.S. Depart- ment of Agriculture-Department of Energy-National Science Foun- dation Plant Science Centers Program and a unit of the Cornell Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation, a consortium of industries, and the U.S. Army Research Office. H.Z.I. was supported by a Peace Fellowship Award from the Egyptian Cultural and Educational Bu- reau.

* Present address: Department of Botany, Duke University, Dur- ham, NC 27706.

Present address: Institute of Graduate Studies and Research, Department of Environmental Studies, University of Alexandria, Alexandria, Egypt.

* Corresponding author; fax 1-607-255-5407.

vation of pyrimidine dimers), excision repair, and damage tolerance by recombination or trans-lesion DNA synthesis (Walker, 1984; McLennan, 1988; Sancar and Sancar, 1988; Sassanfar and Roberts, 1990; Friedberg, 1991). Our knowledge of the corresponding repair pathways in plants, particularly in chloroplasts, is still extremely limited (Small, 1987; McLennan, 1988).

Plants have apparently evolved strategies to prevent DNA damage. The phenylpropanoid pathway, responsible for the synthesis of UV-light protective flavonoids, is induced by UV irradiation (Dangl et al., 1987; Lipphardt et al., 1988; Mc- Lennan, 1988; Hahlbrock and Scheel, 1989). Catalase and superoxide dismutase are induced in response to oxidative stress, which can damage DNA via free radicals (Scandalios, 1990). A variety of conditioning pretreatments can enhance the survival of plants to irradiation or treatments with chemical mutagens (Heindorff et al., 1987; McLennan, 1988). These results were interpreted by the authors as providing evidence for inducible DNA-repair systems in plants, although direct biochemical evidence for such systems is lacking.

Severa1 DNA-repair mechanisms have been demonstrated or inferred in plastids. In Euglena gvacilis chloroplasts, as reported for cyanobacteria (Levine and Thiel, 1987), photoreactivation of DNA is very active (Nicolas et al., 1980), but repair in the dark seems relatively inefficient (Nicolas et al., 1980; Netrawali and Nair, 1984). Photoreactivation and a dark repair process, presumably excision repair, also occur in chloroplasts of Chlamydomonas reinhardtii (Small, 1987). Se- quences homologous to the uvrC gene of Eschevichia col i , part of the uvrABC excision nuclease, have been detected by Southern hybridization in the chloroplast genome of C. reinhardtii (Oppermann et al., 1989). Some enzymic activities potentially involved in an excision repair mechanism are present in chloroplasts of higher plants (Kessler, 1971; Vele- mínsky et al., 1980; McKown and Tewari, 1984; Bensen and Warner, 1987; Nielsen and Tewari, 1988; Mills et al., 1989; Pyke et al., 1989; Cannon and Heinhorst, 1990). However, direct evidence for such a mechanism is still lacking.

An error-prone DNA repair involving recombinational events has been postulated in chloroplasts of E. gracilis (Ni-

Abbreviations: MMC, mitomycin C; MMS, methyl methanesulfonate; NA, nalidixic acid; SSB, Escherichia coli single-stranded DNA- binding protein; ssDNA, single-stranded DNA; TBS, Tris-buffered saline. -

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156 Cerutti et al. Plant Physiol. Vol. 102, 1993

colas et al., 1985). The sequence conservation of the chloroplast genome in higher plants and green algae also suggests an active process of gene conversion, particularly in the large inverted repeats where mutations, insertions, and deletions are fixed symmetrically in both copies (Lemieux et al., 1988; Svab et al., 1990; Boynton et al., 1991; Clegg et al., 1991). Evidence for chloroplast DNA recombination is extensive in the unicellular green alga Chlamydomonas (Lemieux et al., 1988; Boynton et al., 1991). In higher plants, recombination between chloroplast genomes has been demonstrated after protoplast fusion of different genetic lines (Medgyesy et al., 1985; Thanh and Medgyesy, 1989). Moreover, in transformation experiments, homologous donor DNA appears to integrate stably into the plastome by recombination (Svab et al., 1990).

Although very little is known about the enzymology of DNA repair and recombination in chloroplasts, a relationship with eubacterial systems would be expected, considering the endosymbiotic origin of plastids (Gray, 1989). Because of the central role played by the RecA protein in DNA recombination and repair in eubacteria (Walker, 1984; Sancar and Sancar, 1988; Roca and Cox, 1990), we sought to identify a related protein in chloroplasts of higher plants. In addition, we have recently cloned an Arabidopsis thaliana cDNA encoding a protein 53% identical with E. coli RecA, including a region of nonhomology at its amino terminus consistent with a chloroplast transit peptide (Cerutti et al., 1992). Here we report finding a 39-kD protein in chloroplasts of pea (Pisum sativum L.) that is immunologically related to E. coli RecA. It seems to bind ssDNA, and its steady-state leve1 is enhanced by DNA-damaging agents. Studies with protein synthesis inhibitors suggest that this protein is encoded in the nucleus. To our knowledge, these results provide the first biochemical evidence for an inducible DNA repair/recombination system in chloroplasts of higher plants.

MATERIALS A N D METHODS

Protoplast Preparation

Pea seedlings (Pisum sativum L. cv Progress No. 9) were grown as described by Nivison et al. (1986). Unfolded, but not fully expanded, leaves (1 g fresh weight) were surface sterilized with NaOC1, cut into strips, and placed into 9-cm Petri dishes with 10 mL of buffer A (20 mM Mes, pH 5.8; 350 mM sorbitol; 1 mM KH-J'O,; 5 mM MgC12; 30 mM CaCh; 1 mM DTT; 1% [w/v] BSA) containing 3% (w/v) Cellulysin (Calbiochem) and 0.5% (w/v) Macerase (Calbiochem). Dishes were incubated for 4 to 5 h at room temperature in the dark, with shaking at 40 rpm for the final hour. Protoplasts were isolated by filtration through a 100-fim nylon mesh, followed by centrifugation (5 min, 75g). The pellet was resuspended in buffer B (50 mM Tricine, pH 7.0; 380 mM SUC; 20 mM CaC12; 0.5% [w/v] BSA; 25% [v/v] Percoll [Sigma]) and overlaid with buffer C (buffer B without Percoll) and buffer D (buffer C with 380 mM sorbitol instead of SUC). After centrifugation (12 min, 370g), intact protoplasts were col- lected at the interface between buffers C and D. The purified protoplasts were washed once (5 min, 75g) with either chloroplast isolation buffer (Nivison et al., 1986) for cell fraction-

ation or culture medium (Lehminger-Mertens and Jacobsen, 1989) for treatment with DNA-damaging agents. Finally, protoplasts were resuspended in the same solutions, counted, and adjusted to the desired density.

Cell Fractionation

Protoplasts were broken by five passages through a 10-pm nylon mesh. Many nuclei are broken by this treatment, whereas most chloroplasts and mitochondria remain intact. Chloroplasts were isolated by centrifugation of the homogenate on discontinuous Percoll gradients and subfractionated as previously described (Nivison et al., 1986). Mitochondria were purified by isopyknic centrifugation on self-forming Percoll gradients (Skubatz and Bendich, 1990). After organelles were removed from the homogenate by centrifugation (30 min, 12,OOOg), the supernatant was concentrated by acetone precipitation (Nivison et al., 1986) to give the cytosolic/nuclear fraction. The method for protein extraction from the cellular fractions has been described (Nivison et al., 1986).

Western Blot Analysis

Proteins from the different cellular fractions were separated on 12% SDS-polyacrylamide gels and electroblotted onto nitrocellulose (Towbin et al., 1979). The efficiency of transfer was determined by staining with Ponceau S (Harlow and Lane, 1988). Blots were blocked with 5% (w/v) nonfat dry milk in TBS (50 mM Tris-HC1, pH 7.4; 200 mM NaCl) for 1 h at room temperature. RecA antiserum was added in the same solution at a 1:ZOOO dilution and incubated overnight at 4OC with agitation. After three 15-min washes with TBS (at least one containing 0.1% [v/v] Tween ZO), the filters were blocked as before and incubated with a 1:ZOOO dilution of goat anti- rabbit immunoglobulin G conjugated to horseradish peroxidase for 2 to 3 h at room temperature. Blots were then washed three times with 5% (w/v) nonfat dry milk in TBS and three times with TBS (at least once with TBS containing 0.1% [v/v] Tween 20). A chemiluminescent substrate (Amer- sham) was used for the autoradiographic detection. In some cases, the peroxidase was inactivated by incubation in 15% (v/v) H202 for 30 min, and the blots were reprobed with an antibody against the subunit of the chloroplast ATP synthetase. Relative protein amounts were determined by densitometric scanning (Universal Imaging, Inc.) of the autoradiographic signals.

The polyclonal antiserum to E. coli RecA was affinity purified by incubation with a RecA- bacterial lysate bound to nitrocellulose filters (Huynh et al., 1985). The lysate was prepared from E. coli strain JC10289 (Csonka and Clark, 1979). Purified antiserum was assumed to contain 10 mg mL-' of immunoglobulin G (Harlow and Lane, 1988). Ali- quots were incubated with a 10-fold M excess of purified E. coli RecA (United States Biochemical) or BSA (Sigma) overnight at 4OC. These pretreated antisera were used for immunodetection as described above.

Bandshift Gel Assay

ssDNA was prepared from pUC8 plasmids by digestion of one strand with exonuclease I11 and purification of the re-



Chloroplast RecA Homolog lnduction in Pea 157

maining strand on agarose gels (Sambrook et al., 1989). The ssDNA was labeled with T4 polynucleotide kinase and [Y-~~P]ATP (Sambrook et al., 1989). Chloroplasts were isolated from pea leaves as described by Nivison et al. (1986). They were resuspended in incubation buffer (25 mM Hepes- KOH, pH 7.5; 330 mM sorbitol; 20 mM KCl; 3 mM MgCl,; 1 mM DTT; 0.05% [w/v] BSA), at approximately 1.5 to 2.0 mg Chl mL-', and broken by adding Triton X-100 to a final concentration of 0.1% (v/v). Membranes were removed by centrifugation (5 min, 12,OOOg) at 4OC. One hundred-micro- liter aliquots of the supernatant, either native or boiled for 10 min, were incubated with 200 ng of 32P-ssDNA for 30 min at room temperature. One-half of each reaction, adjusted to 10% (v/v) glycerol, was loaded on a nondenaturing agarose gel prepared in modified buffer (25 mM Tris-acetate, pH 7.5; 0.1 mM EDTA). After electrophoresis, the gels were incubated in transfer buffer (Towbin et al., 1979) containing 0.1% (w/ v) SDS for 30 min. Electroblotting and immunodetection were done as described for westem blot analysis. After the chemiluminescent product had decayed, 32P-ssDNA was detected by autoradiography.

In some cases the reactions were run on low-melting-point agarose gels. Gel pieces along the different lanes were cut out and those containing the highest radioactivity identified by measuring Cerenkov radiation in the scintillation counter. Proteins associated with 32P-ssDNA were purified from the agarose slices by a dilute-freeze-spin procedure (as described in the FMC Corp. technical bulletin). Briefly, each gel slice was diluted with extraction buffer (50 mM Tris-HC1, pH 8.0; 1 mM EDTA; 0.1% [w/v] SDS), melted at 7OoC, and mixed thoroughly. This mixture was allowed to solidify on ice and then frozen at -7OOC for 1 to 2 h. After thawing on ice, the mixture was centrifuged in a microfuge (25 min, 12,OOOg). Proteins were recovered from the supernatant by acetone precipitation (Nivison et al., 1986) and analyzed by western blotting.

Southwestern Blot Analysis

Chloroplast proteins were boiled in sample buffer and separated by SDS-PAGE (Nivison et al., 1986). Instead of renaturing the proteins in the gel, followed by capillary blotting to nitrocellulose (Bowen et al., 1980), we electroblotted denatured proteins as described above. The filters were incubated in renaturation buffer (10 mM Tris-HC1, pH 7.5; 50 mM NaCl; 1 mM DTT; 3 mM MgC12; 4 M urea) for 2 h at room temperature, which included three changes of buffer. This step removes SDS and methanol and, presumably, fa- cilitates subsequent renaturation of the proteins. Blots were then washed three times with binding buffer (renaturation buffer without urea) for 2 h at room temperature and blocked with the same buffer containing 5% (w/v) nonfat dry milk. Either a 24-mer or an 18-mer oligonucleotide, labeled with [-p3'P]ATP and T4 polynucleotide kinase (Sambrook et al., 1989), was added in binding buffer containing 0.25% (w/v) nonfat dry milk and 0.05% (v/v) Triton X-100. After 1 h of incubation at room temperature, filters were washed four times with binding buffer for 30 min. Finally, blots were exposed to x-ray film with an intensifying screen.

Protoplast Treatments

Ten milliliters of protoplasts (1 X 106 mL-') were placed in 9-cm Petri dishes. Liquid LP* medium (Lehminger-Mertens and Jacobsen, 1989), without hormones, was the standard culture medium for a11 experiments. Protoplast viability was followed by staining with fluorescein diacetate (Power and Chapman, 1985).

MMS, MMC, or NA was added to the desired final concentrations to individual Petri dishes, and protoplasts were incubated in the dark for the entire experimental period. At the end, intact protoplasts were reisolated by flotation and proteins analyzed by western blotting as described above. When inhibitors of translation were used, they were added to the protoplasts 1 h before the onset of the DNA-damaging treatments. A11 inhibitors and DNA-damaging agents were obtained from Sigma.

RESULTS

Proteins lmmunologically Related to E. coli RecA in Protoplasts of Pea

Proteins from different cellular fractions were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with an antiserum to E. coli RecA. Preimmune serum did not detect any bands on the gels of the plant extracts (data not shown). However, several proteins cross-reacted with the polyclonal antibody raised against E. coli K12 RecA, as shown in Figure 1. Four proteins, ranging from 46.5 to 32.5 kD, were present in the chloroplast fraction (Fig. 1, A and B). They were also visible in the protoplast fraction (Fig. lA, lane 2) after very long exposures (data not shown). A11 of them were soluble proteins localized in the chloroplast stroma (Fig. 1B). The most intense band, with an apparent molecular mass of 39 kD, was very similar in size to E. coli RecA (Fig. 1B). One protein, with an apparent molecular mass of 55 kD, was detected in the mitochondrial fraction (Fig. lA, lane 4), and another protein with an apparent molecular mass of 30 kD, was present in the cytosolic/nuclear fraction (Fig. lA, lane 3). The precise subcellular location of this protein has not been determined, because as described in "Materials and Methods," the cytosolic/nuclear fraction is a mixture of soluble nuclear and cytoplasmic proteins. Similar proteins were also detected in A. thaliana (Cerutti et al., 1992, and data not shown).

Mammalian and yeast cells contain proteins that cross- react to various extents with polyclonal antisera to E. coli RecA (Elledge and Davies, 1987; Hurd et al., 1987; Angulo et al., 1989,1991). However, several genes isolated by screen- ing expression libraries with anti-RecA antibodies were found to encode proteins functionally unrelated to RecA (Elledge and Davies, 1987; Hurd et al., 1987; Angulo et al., 1991). Therefore, we tried to asses the degree of immunological relatedness between the chloroplast proteins and E. coli RecA. The polyclonal antiserum was affinity purified by incubation with a protein extract, bound to nitrocellulose filters from an E. coli strain from which the recA gene was deleted. This treatment removes contaminating antibodies recognizing epitopes in other prokaryotic proteins, distinct from RecA. This purified antiserum was preincubated with a 10-fold M excess




1 2 3 4 5 kD

.-66

-45-36

-29-24

B

Figure 1. Proteins immunologically related to £. coll RecA in pro-toplasts of pea. A, Lane 1, Purified £. coli RecA (the double natureof the band is due to proteolytic degradation); lane 2, protoplastfraction; lane 3, cytosolic/nuclear fraction; lane 4, mitochondrialfraction; lane 5, chloroplast fraction. B, The chloroplast proteinsimmunologically related to E. coli RecA are present in the solublefraction. Lane 1, Purified £. coli RecA; lane 2, stroma; lane 3, wholechloroplasts; lane 4, thylakoids.

of either purified E. coli RecA or BSA, as a control, and usedto identify proteins in the chloroplast fraction (Fig. 2). Thepreincubation with RecA is expected to block the reaction ofthe antibody with authentic RecA-related proteins having thesame epitopes.

Affinity purification of the antiserum with the crude cellextract lacking RecA eliminated detection of the 46.5- and33.5-kD chloroplast proteins, even after very long exposures(Fig. 2A). However, with immunological detection the 39-and 32.5-kD proteins were still clearly visible. On the otherhand, after preincubation of the antibody with E. coli RecA,the 39-kD chloroplast protein was no longer visible (Fig. 2B).As expected, the E. coli RecA loaded as a standard also wasno longer detected by the antibody. However, the 32.5-kDprotein was only slightly affected by this treatment (Fig. 2B).These results suggest that the 46.5-, 33.5-, and 32.5-kDproteins are probably cross-reacting with contaminating an-tibodies (i.e. non-anti-RecA) present in the polyclonal anti-serum. However, the 39-kD chloroplast protein seems spe-cifically related to E. coli RecA.

A Chloroplast Protein Immunologically Related to E. coliRecA Stably Associates with ssDNA

E. coli RecA binds readily to ssDNA in the absence of anucleotide cofactor, and the nucleoprotein filaments are sta-

ble to prolonged incubation (Bryant et al., 1985; Zlotnick etal., 1990). To test whether the 39-kD chloroplast proteinbehaves as RecA with respect to DNA binding, 32P-ssDNAwas incubated with a crude stromal extract from pea chlo-roplasts. As a control, the stromal extract was boiled for 10min before the incubation. The reactions were loaded on anagarose gel and, after electrophoresis, transferred to a nitro-cellulose membrane. Proteins related to E. coli RecA wereimmunologically identified by a chemiluminescent assay. Thesame blot was used to detect the 32P-ssDNA by autoradiog-raphy, after the chemiluminescent product had decayed.

A portion of the proteins cross-reacting with E. coli RecAantiserum were shifted to a higher position in the gel, comi-grating with the similarly shifted 32P-ssDNA, as shown inFigure 3A. When the extract had been boiled no gel retarda-tion could be observed (Fig. 3A). To determine which chlo-roplast protein(s) was associated with the ssDNA, the reac-tions were run on a low-melting-point agarose gel, and theband containing the 32P-ssDNA was excised. Proteins werepurified from the agarose slice, separated by SDS-PAGE,transferred to nitrocellulose, and immunodetected with un-purified anti-RecA polyclonal antibody. Even after very longexposures, the 39-kD chloroplast protein was the only bandvisible on the blots (Fig. 3B). This protein also binds to anssDNA-cellulose column, and it appears to be in a multimericconformation in its native state (data not shown). Theseresults are consistent with the 39-kD protein being a chloro-plast homolog of E. coli RecA.

As shown in Figure 3A, free ssDNA ran off the gel underour electrophoretic conditions. Because some labeled ssDNAremained on the gel after incubation with the boiled extract,we infer that there must be a heat-stable stromal factor(s)binding to it. The further retardation of ssDNA seen with thenative extract is presumably due to binding by the 39-kDchloroplast protein (Fig. 3A). E. coli SSB is extraordinarilystable and can regain full function after being heated at100°C for several minutes (Meyer et al., 1979). An openreading frame of 273 amino acids, with local homology to E.co/i SSB, has been found in the chloroplast genome of tobacco(Shinozaki et al., 1986). In addition, nuclear-encoded ribo-

1 2 3 kD 1 2 3 kD

-39.0

-32.5

-39.0

-32.5

Figure 2. Detection of chloroplast proteins by an affinity-purifiedanti-RecA antiserum. A, Antibody preincubated with BSA. Lane 1,Purified £. coli RecA; lane 2, proteins from a recA" £. col/' strain (JC10289); lane 3, chloroplast proteins. B, Elimination of cross-reac-tivity with the 39-kD chloroplast protein by preincubation ofthe antibody with purified £. co/i RecA. Lanes 1 to 3 are as de-scribed for A.



Chloroplast RecA Homolog Induction in Pea 159

A ssDNA1 2

Protein1 2

-bound- •

free _|̂ ^Hprotein'̂ ^^H

B 1 2 kD C 1 2 3 4 kD

--39.0 -39.0

^••™-

J

•-28.0

Figure 3. Chloroplast proteins immunologically related to E. coliRecA comigrate with ssDNA on agarose gels. A, 32P-ssDNA wasincubated with either boiled or native stromal extracts, and thereactions were separated on agarose gels and transferred to nitro-cellulose filters. 32P-ssDNA was detected by autoradiography(ssDNA), and proteins were identified immunologically (Protein).Lane 1, Boiled stromal extract; lane 2, native stromal extract. B,Proteins associated with ssDNA were purified from the agaroseslice, separated by SDS-PACE, and immunodetected by westernblotting. Lane 1, Purified E. coli RecA; lane 2, proteins isolated fromthe agarose slice. C, Southwestern blot showing ssDNA-bindingproteins in chloroplasts of pea. Lane 1, Purified E. coli RecA; lane2, thylakoidal proteins; lane 3, total Chloroplast proteins; lane 4,stromal proteins.

nucleoproteins of 28 to 30 kD have been purified fromtobacco chloroplasts by their ability to bind ssDNA cellulose(Li and Sugiura, 1990). To explore the possibility that the peastromal extract contains an ssDNA-binding protein tolerantto boiling, we used a modified southwestern procedure, asdescribed in "Materials and Methods." Despite the ratherharsh treatments, a protein with an apparent molecular massof 28 kD was still able to bind ssDNA (Fig. 3C). Purified £.coli RecA, included in the same blot as a control, was inacti-vated completely by the treatments (Fig. 3C). Thus, it istempting to speculate that chloroplasts contain a functionalanalog of the E. coli SSB protein, although of differentmolecular mass, because SSB is a homotetramer of 18.9-kDsubunits (Meyer et al., 1979).

DMA-Damaging Agents Increase the Steady-StateLevel of the 39-kD Chloroplast Protein

DNA repair has been postulated as the primary role ofenzymic system(s) involving RecA in E. coli (Walker, 1984;

Smith and Wang, 1989; Roca and Cox, 1990). Activated RecApromotes the autocatalytic cleavage of the LexA represser,resulting in increased expression of genes involved in DNArepair (including recA itself). In addition, genetic recombina-tion is essential for the repair of certain DNA lesions, likedouble-strand breaks, where an intact complementary stranddoes not exist and the information required for repair mustcome from a separate DNA molecule (Sancar and Sancar,1988; Resnick et al., 1989; Smith and Wang, 1989).

We tested whether the level of the 39-kD Chloroplastprotein was affected by treating pea protoplasts with theDNA-damaging agents MMS, MMC, and NA. Althoughacting in different ways, the chemicals and concentrationsused are expected to cause loss of information on both strandsof DNA. The monofunctional alkylating agent MMS reactswith purine bases and especially attacks the N-7 position ofguanine (Saffhill et al., 1985). However, at the high concen-trations used here, MMS is likely to induce DNA strandbreaks (Mirzayans et al., 1988). MMC is a bifunctional alkyl-ating agent that is activated by reduction and can forminterstrand cross-links (Tomasz et al., 1987; Dusre et al.,1989). NA is an inhibitor of subunit A in bacterial DNAgyrase, a type II topoisomerase. An immunologically relatedenzyme has been detected in chloroplasts (Mills et al., 1989;Pyke et al., 1989). NA stabilizes the linkage between theenzyme and the cut DNA strands, causing a double-strandbreak (Drlica and Franco, 1988).

Treatment of pea protoplasts with these reagents enhancedthe steady-state level of the 39-kD Chloroplast protein im-munologically related to E. coli RecA (Fig. 4). Preliminaryresults showing increased levels of the Chloroplast proteininduced by MMC were reported previously (Cerutti et al.,1992). The chloroplast 39-kD protein reached a maximumlevel after approximately 12 to 15 h in the presence of MMS(Fig. 5A). The degree of induction varied with the DNA-damaging agent, the concentration used, and the duration ofexposure (Figs. 4 and 5A). Under optimal inducing conditions,the level of the 39-kD chloroplast protein increased 3.5-fold(Fig. 4).

Induction of the putative RecA homolog seems specific,because most proteins in a Coomassie blue-stained gelshowed no change compared with the control from proto-plasts not exposed to a DNA-damaging agent (data notshown). We also reprobed the nitrocellulose filters with anantibody against the y subunit of the chloroplast ATP syn-thetase, a nuclear-encoded protein similar in size to E. coliRecA. As expected, this protein was not induced by DNA-damaging agents and served as a control for the properloading of the lanes (Fig. 5B). Another indication of a specificeffect is that the 30-kD protein in the cytosolic/nuclear frac-tion was induced with different kinetics by MMS (Fig. 5A),and it was not affected by treatments with NA or MMC (datanot shown). The viability of the protoplasts, as determinedby fluorescein diacetate staining, was not significantly af-fected by any of the treatments (data not shown).

The 39-kD Chloroplast Protein Appears to beEncoded in the Nucleus

We used protein synthesis inhibitors to gain preliminaryinformation concerning the site of synthesis of the protein




30 7000

NA MMC MMSCONCENTRATION IN nM

Figure 4. The steady-state level of the 39-kD chloroplast protein isenhanced by DMA-damaging agents. Pea protoplasts were incu-bated for 12 to 15 h in the presence of the different chemicals. The39-kD chloroplast protein was detected by western blotting, andrelative protein amounts were determined by densitometric scan-ning of the autoradiographic films. Bars indicate mean proteinamount ± SE (n = 3). C, Control.

whose level increases in response to DNA-damaging agents.Erythromycin and chloramphenicol are inhibitors of transla-tion by prokaryotic type (70S) ribosomes (Gale et al., 1981).Erythromycin has been shown to inhibit protein synthesisexclusively in chloroplasts of plant cells, whereas chloram-phenicol affected both chloroplastic and, less effectively,mitochondria! protein synthesis (Tassi et al., 1983; Sasaki andKuroiwa, 1988). Anisomycin and emetine inhibit translationon eukaryotic type (80S) ribosomes, apparently with higherspecificity than cycloheximide (Gale et al., 1981; Schmidtet al., 1983).

The increase in the steady-state level of the 39-kD protein,in response to MMS was prevented by emetine and aniso-mycin (Fig. 6). However, chloramphenicol and erythromycinlacked significant effect. The simplest interpretation of theseresults is that the induction involves de novo protein synthe-sis on eukaryotic type ribosomes. This also suggests that the39-kD chloroplast protein is encoded in the nucleus.

and Wang, 1989; Roca and Cox, 1990; Sassanfarand Roberts,1990). According to the serial endosymbiosis theory, chloro-plasts arose by symbiotic association between ancestral eu-karyotes and free-living cyanobacterial progenitors (Gray,1989). The key role played by RecA in DNA metabolism inmost eubacteria suggested that such a protein might also bepresent in chloroplasts.

We found here a protein in pea chloroplasts recognized bya polyclonal antiserum to E. coli K12 RecA. This cross-reactingprotein has an apparent molecular mass (39 kD) very similarto that of E. coli RecA. The recognition of this protein waseliminated by preincubation of the antibody with purified E.coli RecA, indicating that the two proteins have commonepitopes. However, a subunit of yeast ribonucleotide reduc-tase (Elledge and Davis, 1987; Hurd et al., 1987) and themouse DNA-binding protein KIN17 (Angulo et al., 1991) alsoshare antigenic determinants with E. coli RecA, although theyare functionally unrelated. Although we cannot rule out sucha possibility here, the evidence discussed below stronglysuggests that the 39-kD chloroplast protein is a homolog ofE. coli RecA.

Using a combination of bandshift gel assay and westernblotting, we were able to show that the 39-kD chloroplastprotein comigrates with ssDNA. This suggests that the 39-kD protein binds directly to ssDNA, as expected for a RecAhomolog. Secondary binding of the 39-kD protein to a pri-mary ssDNA-binding protein is possible but much less likely.We have also found an ATP-dependent strand-transfer activ-ity, an essential function of RecA, in crude stromal extractsfrom pea (Cerutti and Jagendorf, 1993). Finally, we have

Time (h)6 12 18 24 kD

-39.0

-30.0

DISCUSSION

Although many organisms have been shown to possessinducible DNA repair/recombination systems (Walker, 1984;McLennan, 1988; Sancar and Sancar, 1988; Friedberg, 1991),very little has been done to investigate possible mechanismsin chloroplasts. RecA of £. coli is a multifunctional enzymerequired for homologous recombination, induction of theSOS system in response to DNA damage, postreplicativerepair, SOS mutagenesis, and recovery of DNA synthesisafter damage (Walker, 1984; Sancar and Sancar, 1988; Smith

B'-38.0

Figure 5. Time course of induction of the 39-kD chloroplast proteinby the addition of MMS (7 FTIM) to pea protoplasts. A, Probing withantiserum to E. coli RecA. Lanes indicate time after the onset oftreatment. B, The same filter shown in A was reprobed with anti-serum to the 7 subunit of the chloroplast ATP synthetase as acontrol for the proper loading of the lanes.



Chloroplast RecA Homolog Induction in Pea 161

c1

**

Ch2

Er3

I

4

An5 6

Em7 8

R9

.1»kD

-39.0

Figure 6. Effect of protein synthesis inhibitors on the induction ofthe 39-kD chloroplast protein by MMS. MMS (7 ITIM) was added 1h after the inhibitors, and the protoplasts were incubated foranother 12 h before protein isolation and electrophoresis. Lane 1,Noninduced control; lane 2, chloramphenicol (100 Mg rnl_~'); lane3, erythromycin (300 \i% ml"1); lane 4, induced control; lane 5,anisomycin (100 jig ml"1); lane 6, anisomycin (300 ng ml"1); lane7, emetine (1.0 mg ml"1); lane 8, emetine (2.5 mg mL~'); lane 9,purified £. coli RecA.

cloned an A. thaliana cDNA that, except for a predictedchloroplast transit peptide, encodes a protein highly homol-ogous to £. coli RecA (Cerutti et al., 1992).

DNA repair has been considered the primary function ofhomologous recombination in maintaining cell viability, andthis would be the most essential role of RecA in E. coli(Walker, 1984; Resnick et al., 1989; Smith and Wang, 1989;Roca and Cox, 1990). Therefore, we tested the effect of MMS,MMC, and NA on the steady-state level of the chloroplastcross-reacting protein. The chemicals were used at concentra-tions causing DNA lesions that, presumably, could be re-paired by a recombinational pathway (Tomasz et al., 1987;Drlica and Franco, 1988; Mirzayans et al., 1988; Dusre et al.,1989; Resnick et al., 1989; Smith and Wang, 1989). Theamount of the 39-kD protein increased in response to theseagents, reaching a maximum approximately 12 to 15 h afterthe onset of treatments. Similar kinetics of induction havebeen found for nuclear-encoded enzymes of the phenylpro-panoid pathway, in response to UV irradiation (Dangl et al.,1987; Lipphardt et al., 1988; Hahlbrock and Scheel, 1989)and, for catalase and superoxide dismutase, in response tooxidative stress (Scandalios, 1990).

The response of pea protoplasts to DNA-damaging agentsseems specific, rather than a general reaction to stress, be-cause the level of the 39-kD protein was not affected bywounding during protoplast isolation (data not shown). Sim-ilarly, isolated parsley protoplasts retain specific responsive-ness to treatments inducing chalcone synthase (Dangl et al.,1987; Lipphardt et al., 1988). There is a large body of evi-dence implicating DNA as a primary target in the cytotoxicityof MMS and MMC (Saffhill et al., 1985; Dusre et al., 1989).NA was shown to induce DNA strand breaks in isolated peachloroplasts (Mills et al., 1989). However, it can also actas an uncoupler of ATP synthesis by both mitochondriaand chloroplasts, particularly at concentrations greater than400 MM (Gallagher et al., 1986; Mills et al., 1989). If denovo protein synthesis is involved in the induction of the39-kD protein (see below), a reduction in ATP productionwould diminish the magnitude of the response. Because wedetected an inductive effect of NA, the interference withenergy production could not have been overriding. Thus, theresults with the three DNA-damaging agents are consistentwith a role for the 39-kD chloroplast protein in DNA repair/recombination.

Antibiotics preventing chloroplast translation (erythromy-

cin and chloramphenicol) did not affect the induction of the39-kD chloroplast protein after exposure to MMS. However,the 80S ribosomal inhibitors emetine and anisomycin pre-vented this response. The concentrations of erythromycin,chloramphenicol, and anisomycin were similar to those pre-viously used in several plant cell systems (Schmidt et al.,1983; Tassi et al., 1983; Sasaki and Kuroiwa, 1988). Emetine,however, was used at a concentration about 8-fold higherthan that required to inhibit protein synthesis in animalsystems. At these concentrations it was found to precipitatesome proteins from crude cell extracts of bean (Jones andNorthcote, 1981). Therefore, it is not clear whether its effectwas completely specific.

Nevertheless, the simplest interpretation of our data is thatthe increase in the antigen involves de novo protein synthesison 80S ribosomes, and, therefore, the 39-kD chloroplastprotein is most likely encoded in the nucleus. In support ofthis interpretation, the time course of induction is similar tothose of other nuclear-encoded enzymes, as noted above.

An alternative possibility might be posttranslational stabi-lization of the 39-kD protein in response to DNA-damagingagents, as reported for the p53 cellular tumor antigen (Maltz-man and Czyzyk, 1984), rather than de novo synthesis.Although we have no direct evidence to rule out this possi-bility, posttranslational stabilization was found to be muchfaster (in mouse cells) than the response observed here, witha significant effect immediately after the onset of treatments(Maltzman and Czyzyk, 1984).

Hybridization results also support a nuclear localization fora gene encoding a RecA homolog. Sequences homologous tothe Synechococcus recA gene were found in Southern blotsusing total DNA but not in those using chloroplast or mito-chondria! DNA from pea (Cerutti et al., 1992, and data notshown). These results, together with identification of a ge-nomic clone for the RecA-like protein (M.-N. Binet, M. Os-man, and A.T. Jagendorf, unpublished data) show that theA. thaliana recA gene, selected using a cyanobacterial probe(Cerutti et al., 1992), is located in the nucleus.

The body of evidence to date is most consistent with the39-kD chloroplast protein being a homolog of E. coli RecA.It also supports the relationship between the chloroplast DNArepair/recombination system and its eubacterial counterpart.However, unique aspects derived from the nucleo-cyto-plasmic interaction are also revealed. The recA gene is nowlocated in the nucleus of an eukaryote (Cerutti et al., 1992)and subject to a different regulatory system. Recently, an-other A. thaliana cDNA was isolated by complementation ofan E. coli strain deficient in DNA repair (Ap/irA«i>rBArecA).It encodes a putative chloroplast protein with homology toparts of £. coli recA and yeast RAD genes (Pang et al., 1992).Further research should reveal whether a chloroplastic SOS-like system exists and whether other chloroplast enzymes,involved in DNA repair/recombination, are induced in re-sponse to DNA damage. The regulatory mechanism(s) con-trolling the response of the 39-kD chloroplast protein toDNA-damaging agents is also of interest.

ACKNOWLEDGMENT

We would like to thank J. W. Roberts for providing antibodies toE. co/i RecA.




Received October 12, 1992; accepted January 29, 1993. Copyright Clearance Center: 0032-0889/93/l02/0l55/09.

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of Pea (Pisum sativum 1.) Protoplasts with DNA- Damaging ...Plant Physiol. (1993) 102: 155-163 Treatment of Pea (Pisum sativum 1.)Protoplasts with DNA- Damaging Agents lnduces a 39-Kilodalton