Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Jun;15(6):3003–3011. doi: 10.1128/mcb.15.6.3003

Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA.

H Cerutti 1, A M Johnson 1, J E Boynton 1, N W Gillham 1
PMCID: PMC230531  PMID: 7760798

Abstract

The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance. The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart. Therefore, we examined whether dominant negative mutants of the E. coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination. These results strongly support the existence of a RecA-mediated recombination system in chloroplasts. We also found that the wild-type E. coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.

Full Text

The Full Text of this article is available as a PDF (784.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. André C., Levy A., Walbot V. Small repeated sequences and the structure of plant mitochondrial genomes. Trends Genet. 1992 Apr;8(4):128–132. doi: 10.1016/0168-9525(92)90370-J. [DOI] [PubMed] [Google Scholar]
  2. Asai T., Bates D. B., Kogoma T. DNA replication triggered by double-stranded breaks in E. coli: dependence on homologous recombination functions. Cell. 1994 Sep 23;78(6):1051–1061. doi: 10.1016/0092-8674(94)90279-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Asai T., Kogoma T. D-loops and R-loops: alternative mechanisms for the initiation of chromosome replication in Escherichia coli. J Bacteriol. 1994 Apr;176(7):1807–1812. doi: 10.1128/jb.176.7.1807-1812.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bauer C. E., Bollivar D. W., Suzuki J. Y. Genetic analyses of photopigment biosynthesis in eubacteria: a guiding light for algae and plants. J Bacteriol. 1993 Jul;175(13):3919–3925. doi: 10.1128/jb.175.13.3919-3925.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bennett C. B., Lewis A. L., Baldwin K. K., Resnick M. A. Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5613–5617. doi: 10.1073/pnas.90.12.5613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bi X., Liu L. F. recA-independent and recA-dependent intramolecular plasmid recombination. Differential homology requirement and distance effect. J Mol Biol. 1994 Jan 14;235(2):414–423. doi: 10.1006/jmbi.1994.1002. [DOI] [PubMed] [Google Scholar]
  7. Boynton J. E., Gillham N. W., Harris E. H., Hosler J. P., Johnson A. M., Jones A. R., Randolph-Anderson B. L., Robertson D., Klein T. M., Shark K. B. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science. 1988 Jun 10;240(4858):1534–1538. doi: 10.1126/science.2897716. [DOI] [PubMed] [Google Scholar]
  8. Cazaux C., Mazard A. M., Defais M. Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele. Mol Gen Genet. 1993 Aug;240(2):296–301. doi: 10.1007/BF00277070. [DOI] [PubMed] [Google Scholar]
  9. Cerutti H., Ibrahim H. Z., Jagendorf A. T. Treatment of pea (Pisum sativum L.) protoplasts with DNA-damaging agents induces a 39-kilodalton chloroplast protein immunologically related to Escherichia coli RecA. Plant Physiol. 1993 May;102(1):155–163. doi: 10.1104/pp.102.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cerutti H., Jagendorf A. T. DNA Strand-Transfer Activity in Pea (Pisum sativum L.) Chloroplasts. Plant Physiol. 1993 May;102(1):145–153. doi: 10.1104/pp.102.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cerutti H., Osman M., Grandoni P., Jagendorf A. T. A homolog of Escherichia coli RecA protein in plastids of higher plants. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8068–8072. doi: 10.1073/pnas.89.17.8068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clark A. J., Sandler S. J. Homologous genetic recombination: the pieces begin to fall into place. Crit Rev Microbiol. 1994;20(2):125–142. doi: 10.3109/10408419409113552. [DOI] [PubMed] [Google Scholar]
  13. Collin S., Ellis T. H. Evidence for the presence of hairpin chloroplast DNA molecules in barley cultivars. Curr Genet. 1991 Aug;20(3):253–258. doi: 10.1007/BF00326240. [DOI] [PubMed] [Google Scholar]
  14. Cox M. M. Relating biochemistry to biology: how the recombinational repair function of RecA protein is manifested in its molecular properties. Bioessays. 1993 Sep;15(9):617–623. doi: 10.1002/bies.950150908. [DOI] [PubMed] [Google Scholar]
  15. Feng W. Y., Lee E. H., Hays J. B. Recombinagenic processing of UV-light photoproducts in nonreplicating phage DNA by the Escherichia coli methyl-directed mismatch repair system. Genetics. 1991 Dec;129(4):1007–1020. doi: 10.1093/genetics/129.4.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Foster P. L. Adaptive mutation: the uses of adversity. Annu Rev Microbiol. 1993;47:467–504. doi: 10.1146/annurev.mi.47.100193.002343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fox M. S., Radicella J. P., Yamamoto K. Some features of base pair mismatch repair and its role in the formation of genetic recombinants. Experientia. 1994 Mar 15;50(3):253–260. doi: 10.1007/BF01924008. [DOI] [PubMed] [Google Scholar]
  18. Freitag N., McEntee K. Affinity chromatography of RecA protein and RecA nucleoprotein complexes on RecA protein-agarose columns. J Biol Chem. 1988 Dec 25;263(36):19525–19534. [PubMed] [Google Scholar]
  19. Goldschmidt-Clermont M. Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucleic Acids Res. 1991 Aug 11;19(15):4083–4089. doi: 10.1093/nar/19.15.4083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hall R. D., Rouwendal G. J., Krens F. A. Asymmetric somatic cell hybridization in plants. I. The early effects of (sub)lethal doses of UV and gamma radiation on the cell physiology and DNA integrity of cultured sugarbeet (Beta vulgaris L.) protoplasts. Mol Gen Genet. 1992 Aug;234(2):306–314. [PubMed] [Google Scholar]
  21. Harada T., Ishikawa R., Niizeki M., Saito K. Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth. Mol Gen Genet. 1992 May;233(1-2):145–150. doi: 10.1007/BF00587572. [DOI] [PubMed] [Google Scholar]
  22. Harris R. S., Longerich S., Rosenberg S. M. Recombination in adaptive mutation. Science. 1994 Apr 8;264(5156):258–260. doi: 10.1126/science.8146657. [DOI] [PubMed] [Google Scholar]
  23. Horii T., Ogawa T., Ogawa H. Organization of the recA gene of Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jan;77(1):313–317. doi: 10.1073/pnas.77.1.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Horii T., Ozawa N., Ogawa T., Ogawa H. Inhibitory effects of N- and C-terminal truncated Escherichia coli recA gene products on functions of the wild-type recA gene. J Mol Biol. 1992 Jan 5;223(1):105–114. doi: 10.1016/0022-2836(92)90719-z. [DOI] [PubMed] [Google Scholar]
  25. Hosler J. P., Wurtz E. A., Harris E. H., Gillham N. W., Boynton J. E. Relationship between Gene Dosage and Gene Expression in the Chloroplast of Chlamydomonas reinhardtii. Plant Physiol. 1989 Oct;91(2):648–655. doi: 10.1104/pp.91.2.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Khidhir M. A., Casaregola S., Holland I. B. Mechanism of transient inhibition of DNA synthesis in ultraviolet-irradiated E. coli: inhibition is independent of recA whilst recovery requires RecA protein itself and an additional, inducible SOS function. Mol Gen Genet. 1985;199(1):133–140. doi: 10.1007/BF00327522. [DOI] [PubMed] [Google Scholar]
  27. Kolodner R. D., Tewari K. K. Chloroplast DNA from higher plants replicates by both the Cairns and the rolling circle mechanism. Nature. 1975 Aug 28;256(5520):708–711. doi: 10.1038/256708a0. [DOI] [PubMed] [Google Scholar]
  28. Kolodner R., Fishel R. A., Howard M. Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli. J Bacteriol. 1985 Sep;163(3):1060–1066. doi: 10.1128/jb.163.3.1060-1066.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kowalczykowski S. C., Eggleston A. K. Homologous pairing and DNA strand-exchange proteins. Annu Rev Biochem. 1994;63:991–1043. doi: 10.1146/annurev.bi.63.070194.005015. [DOI] [PubMed] [Google Scholar]
  30. Kunz B. A. Thymineless mutagenesis in bacteria. Basic Life Sci. 1985;31:189–209. doi: 10.1007/978-1-4613-2449-2_12. [DOI] [PubMed] [Google Scholar]
  31. Lauder S. D., Kowalczykowski S. C. Negative co-dominant inhibition of recA protein function. Biochemical properties of the recA1, recA13 and recA56 proteins and the effect of recA56 protein on the activities of the wild-type recA protein function in vitro. J Mol Biol. 1993 Nov 5;234(1):72–86. doi: 10.1006/jmbi.1993.1564. [DOI] [PubMed] [Google Scholar]
  32. Lers A., Heifetz P. B., Boynton J. E., Gillham N. W., Osmond C. B. The carboxyl-terminal extension of the D1 protein of photosystem II is not required for optimal photosynthetic performance under CO2- and light-saturated growth conditions. J Biol Chem. 1992 Sep 5;267(25):17494–17497. [PubMed] [Google Scholar]
  33. Louarn J. M., Louarn J., François V., Patte J. Analysis and possible role of hyperrecombination in the termination region of the Escherichia coli chromosome. J Bacteriol. 1991 Aug;173(16):5097–5104. doi: 10.1128/jb.173.16.5097-5104.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mahan M. J., Roth J. R. Role of recBC function in formation of chromosomal rearrangements: a two-step model for recombination. Genetics. 1989 Mar;121(3):433–443. doi: 10.1093/genetics/121.3.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mirzayans R., Liuzzi M., Paterson M. C. Methylmethanesulfonate-induced DNA damage and its repair in cultured human fibroblasts: normal rates of induction and removal of alkali-labile sites in xeroderma pigmentosum (group A) cells. Carcinogenesis. 1988 Dec;9(12):2257–2263. doi: 10.1093/carcin/9.12.2257. [DOI] [PubMed] [Google Scholar]
  36. Modrich P. Mechanisms and biological effects of mismatch repair. Annu Rev Genet. 1991;25:229–253. doi: 10.1146/annurev.ge.25.120191.001305. [DOI] [PubMed] [Google Scholar]
  37. Newman S. M., Harris E. H., Johnson A. M., Boynton J. E., Gillham N. W. Nonrandom distribution of chloroplast recombination events in Chlamydomonas reinhardtii: evidence for a hotspot and an adjacent cold region. Genetics. 1992 Oct;132(2):413–429. doi: 10.1093/genetics/132.2.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nicolas A., Petes T. D. Polarity of meiotic gene conversion in fungi: contrasting views. Experientia. 1994 Mar 15;50(3):242–252. doi: 10.1007/BF01924007. [DOI] [PubMed] [Google Scholar]
  39. Ogawa T., Shinohara A., Ogawa H., Tomizawa J. Functional structures of the recA protein found by chimera analysis. J Mol Biol. 1992 Aug 5;226(3):651–660. doi: 10.1016/0022-2836(92)90622-q. [DOI] [PubMed] [Google Scholar]
  40. Pang Q., Hays J. B., Rajagopal I. Two cDNAs from the plant Arabidopsis thaliana that partially restore recombination proficiency and DNA-damage resistance to E. coli mutants lacking recombination-intermediate-resolution activities. Nucleic Acids Res. 1993 Apr 11;21(7):1647–1653. doi: 10.1093/nar/21.7.1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Piskur J. Inheritance of the yeast mitochondrial genome. Plasmid. 1994 May;31(3):229–241. doi: 10.1006/plas.1994.1025. [DOI] [PubMed] [Google Scholar]
  42. Sancar A., Stachelek C., Konigsberg W., Rupp W. D. Sequences of the recA gene and protein. Proc Natl Acad Sci U S A. 1980 May;77(5):2611–2615. doi: 10.1073/pnas.77.5.2611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sedgwick S. G., Goodwin P. A. Differences in mutagenic and recombinational DNA repair in enterobacteria. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4172–4176. doi: 10.1073/pnas.82.12.4172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Skarstad K., Boye E. Degradation of individual chromosomes in recA mutants of Escherichia coli. J Bacteriol. 1993 Sep;175(17):5505–5509. doi: 10.1128/jb.175.17.5505-5509.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Story R. M., Bishop D. K., Kleckner N., Steitz T. A. Structural relationship of bacterial RecA proteins to recombination proteins from bacteriophage T4 and yeast. Science. 1993 Mar 26;259(5103):1892–1896. doi: 10.1126/science.8456313. [DOI] [PubMed] [Google Scholar]
  46. Story R. M., Weber I. T., Steitz T. A. The structure of the E. coli recA protein monomer and polymer. Nature. 1992 Jan 23;355(6358):318–325. doi: 10.1038/355318a0. [DOI] [PubMed] [Google Scholar]
  47. Suzuki J. Y., Bauer C. E. Light-independent chlorophyll biosynthesis: involvement of the chloroplast gene chlL (frxC). Plant Cell. 1992 Aug;4(8):929–940. doi: 10.1105/tpc.4.8.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Willetts N. S., Clark A. J. Characteristics of some multiply recombination-deficient strains of Escherichia coli. J Bacteriol. 1969 Oct;100(1):231–239. doi: 10.1128/jb.100.1.231-239.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Witkin E. M., Roegner-Maniscalco V., Sweasy J. B., McCall J. O. Recovery from ultraviolet light-induced inhibition of DNA synthesis requires umuDC gene products in recA718 mutant strains but not in recA+ strains of Escherichia coli. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6805–6809. doi: 10.1073/pnas.84.19.6805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Woelfle M. A., Thompson R. J., Mosig G. Roles of novobiocin-sensitive topoisomerases in chloroplast DNA replication in Chlamydomonas reinhardtii. Nucleic Acids Res. 1993 Sep 11;21(18):4231–4238. doi: 10.1093/nar/21.18.4231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wurtz E. A., Boynton J. E., Gillham N. W. Perturbation of chloroplast DNA amounts and chloroplast gene transmission in Chlamydomonas reinhardtii by 5-fluorodeoxyuridine. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4552–4556. doi: 10.1073/pnas.74.10.4552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zweifel S. G., Fangman W. L. A nuclear mutation reversing a biased transmission of yeast mitochondrial DNA. Genetics. 1991 Jun;128(2):241–249. doi: 10.1093/genetics/128.2.241. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES