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Recombination function and recombination kinetics of Escherichia coli single-stranded DNA-binding protein

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  • Life & Medical Sciences
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Science Bulletin

Abstract

It is unknown whether the ssDNA-binding-protein (SSB) possesses the ability to catalyze DNA recombination. We investigated the recombination function of SSB and the recombination kinetics of Escherichia coli using a new transformation method with a modified double-layered plate. We found that SSB catalysed intermolecular recombination in vitro. Its intermolecular recombination rate versus substrate concentration or homologous sequence length fitted the Hill equation, and while the plasmid intramolecular recombination rate versus substrate concentration fitted a positively linear correlation, the dominant intermolecular recombination was a non-homologous recombination in vivo, similar to RecA. However, ssb-dependent recombination occurred later and at a lower recombination rate than the recA-dependent, probably because ssb expression was about two-fold lower than recA during the E. coli earlier growth stage. The affinity to substrate and the recombination efficiency of SSB was lower than RecA, whereas SSB enhanced the catalytic efficiency of RecA. Knocking out both recA and ssb led to loss of recombination. Our results confirmed that as SSB has the recombination function itself as an allosteric enzyme, recA-independent recombination in E. coli should be ssb-dependent. ssb-dependent recombination may be the third DNA double-strand break repair pathway, in addition to recA-dependent recombination and non-homologous end joining.

摘要

单链DNA连接蛋白(SSB)是否具有催化DNA重组的功能尚未见报道。我 们研究了大肠杆菌的SSB 的重组功能, 并用修改的双层平板法研究了大肠杆菌的重 组动力学。我们发现, SSB 能够在体外催化分子间的重组。大肠杆菌 recA 突变株中, 分子间重组速率与底物浓度的关系、或与同源序列长度的关系, 符合 Hill 方程; 质 粒分子内重组的速率与底物浓度呈正相关, 均与 ssb 突变株相同。然而, recA 突变 株发生重组的时间比 ssb 突变株推迟, 且重组速率、对底物的亲和力和重组效率, 均比 ssb 突变株低。同时敲除recA 和 ssb, 大肠杆菌完全失去重组功能。另外还发 现, 在大肠杆菌 recA 突变株、ssb 突变株或非突变株中, 绝大多数重组都是非同源 重组。在大肠杆菌生长的早期, ssb 的表达量比 recA 约低2 倍。结果表明, SSB 能 够独立催化 DNA 重组, 是双链DNA断裂的同源重组修复和非同源重组末端连接修 复二种途径之外的第三种重组修复途径, 即 ssb 依赖的重组修复途径。

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References

  1. Michel B, Grompone G, Flore‘s MJ et al (2004) Multiple pathways process stalled replication forks. Proc Natl Acad Sci USA 101:12783–12788

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  2. Rocha EPC, Cornet E, Michel B (2005) Comparative and evolutionary analysis of the bacterial homologous recombination systems. PLoS Genet 1:e15

    Article  PubMed  PubMed Central  Google Scholar 

  3. Jasin M, Schimmel P (1984) Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments. J Bacteriol 159:783–786

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Zhang Y, Muyrers JP, Testa G et al (2000) DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol 18:1314–1317

    Article  CAS  PubMed  Google Scholar 

  5. Sharan SK, Thomason LC, Kuznetsov SG et al (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4:206–223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bell JC, Plank JL, Dombrowski CC et al (2012) Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA. Nature 491:274–278

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chai R, Qiu C, Liu D et al (2013) β-Glucan synthase gene overexpression and β-glucans overproduction in Pleurotus ostreatus using promoter swapping. PLoS ONE 8:e61693

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Slade D, Radman M (2011) Oxidative stress resistance in Deinococcus radiodurans. Microbiol Mol Biol Rev 75:133–191

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dillingham MS, Kowalczykowski SC (2008) RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiol Mol Biol Rev 72:642–671

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Smith GR (2012) How RecBCD enzyme and Chi promote DNA break repair and recombination: a molecular biologist’s view. Microbiol Mol Biol Rev 76:217–228

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Handa N, Morimatsu K, Lovett ST et al (2009) Reconstitution of initial steps of dsDNA break repair by the RecF pathway of E. coli. Genes Dev 23:1234–1245

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kowalczykowski SC, Dixon DA, Eggleston AK et al (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev 58:401–465

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Forget AL, Kowalczykowski SC (2012) Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search. Nature 482:423–427

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Howard-Flanders P, Theriot L (1966) Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination. Genetics 53:1137–1150

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Fishel RA, James AA, Kolodner R (1981) recA-independent general genetic recombination of plasmids. Nature 294:184–186

    Article  ADS  CAS  PubMed  Google Scholar 

  16. James AA, Morrison PT, Kolodner R (1982) Genetic recombination of bacterial plasmid DNA. Analysis of the effect of recombination-deficient mutations on plasmid recombination. J Mol Biol 160:411–430

    Article  CAS  PubMed  Google Scholar 

  17. Cohen A, Laban A (1983) Plasmidic recombination in Escherichia coli K12: the role of the recF gene function. Mol Gen Genet 189:471–474

    Article  CAS  PubMed  Google Scholar 

  18. Kolodner R, Fishel RA, Howard H (1985) Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli. J Bacteriol 163:1060–1066

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Fu H, Le S, Chen H, Muniyappa K et al (2013) Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein. Nucleic Acids Res 41:924–932

    Article  CAS  PubMed  Google Scholar 

  20. Ragunathan K, Liu C, Ha T (2012) RecA filament sliding on DNA facilitates homology search. Elife 1:e00067

    Article  PubMed  PubMed Central  Google Scholar 

  21. Doherty MJ, Morrison PT, Kolodner R (1983) Genetic recombination of bacterial plasmid DNA. Physical and genetic analysis of the products of plasmid recombination in Escherichia coli. J Mol Biol 167:539–560

    Article  CAS  PubMed  Google Scholar 

  22. Bubeck P, Winkler M, Bautsch W (1993) Rapid cloning by homologous recombination in vivo. Nucleic Acids Res 21:3601–3602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Oliner JD, Kinzler KW, Vogelstein B (1993) In vivo cloning of PCR products in E.coli. Nucleic Acids Res 215:192–5197

    Google Scholar 

  24. Martin A, Toselli E, Rosier MF et al (1995) Rapid and high efficiency site-directed mutagenesis by improvement of the homologous recombination technique. Nucleic Acids Res 23:1642–1643

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Conley EC, Saunders JR (1984) Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli. Mol Gen Genet 194:211–218

    Article  CAS  PubMed  Google Scholar 

  26. Abastado JP, Darche S, Godeau F et al (1987) Intramolecular recombination between partially homologous sequences in Escherichia coli and Xenopus laevis oocytes. Proc Natl Acad Sci USA 84:6496–6500

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bi X, Liu LF (1994) recA-independent and recA-dependent intramolecular plasmid recombination. Differential homology requirement and distance effect. J Mol Biol 235:414–423

    Article  CAS  PubMed  Google Scholar 

  28. Bi X, Lyu YL, Liu LF (1995) Specific stimulation of recA-independent plasmid recombination by a DNA sequence at a distance. J Mol Biol 247:890–902

    Article  CAS  PubMed  Google Scholar 

  29. Morag AS, Saveson CJ, Lovett ST (1999) Expansion of DNA repeats in Escherichia coli: effects of recombination and replication functions. J Mol Biol 289:21–27

    Article  CAS  PubMed  Google Scholar 

  30. Troester H, Bub S, Hunziker A et al (2000) Stability of DNA repeats in Escherichia coli dam mutant strains indicates a Dam methylation-dependent DNA deletion process. Gene 25:95–108

    Article  Google Scholar 

  31. Ribeiro SC, Oliveira PH, Prazeres DM et al (2008) High frequency plasmid recombination mediated by 28 bp direct repeats. Mol Biotechnol 40:252–260

    Article  CAS  PubMed  Google Scholar 

  32. Lovett ST, Gluckman TJ, Simon PJ et al (1994) Recombination between repeats in Escherichia coli by a recA-independent, proximity-sensitive mechanism. Mol Gen Genet 245:294–300

    Article  CAS  PubMed  Google Scholar 

  33. Paytubi S, McMahon SA, Graham S et al (2012) Displacement of the canonical single-stranded DNA-binding protein in the Thermoproteales. Proc Natl Acad Sci USA 109:E398–E405

    Article  ADS  CAS  PubMed  Google Scholar 

  34. Sigal N, Delius H, Kornberg T et al (1972) A DNA-unwinding protein isolated from Escherichia coli: its interaction with DNA and with DNA polymerases. Proc Natl Acad Sci USA 69:3537–3541

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Christiansen C, Baldwin RL (1977) Catalysis of DNA reassociation by the Escherichia coli DNA binding protein: a polyamine-dependent reaction. J Mol Biol 115:441–454

    Article  CAS  PubMed  Google Scholar 

  36. Meyer RR, Laine PS (1990) The single-stranded DNA-binding protein of Escherichia coli. Microbiol Rev 54:342–380

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Lohman TM, Ferrari ME (1994) Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities. Annu Rev Biochem 63:527–570

    Article  CAS  PubMed  Google Scholar 

  38. Marceau AH (2012) Functions of single-strand DNA-binding proteins in DNA replication, recombination, and repair. In: Keck JL (eds) Single-stranded DNA binding proteins: methods and protocols. Methods in molecular biology, vol 922, Humana Press, Totowa, pp 1–21

  39. Roy R, Kozlov AG, Lohman TM et al (2009) SSB protein diffusion on single-stranded DNA stimulates RecA filament formation. Nature 461:1092–1097

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  40. Shen P, Huang HV (1986) Homologous recombination in Escherichia coli: dependence on substrate length and homology. Genetics 112:441–457

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Qiu L, Huang D, Chen CY et al (2008) Severe tuberculosis induces unbalanced up-regulation of gene networks and overexpression of IL-22, MIP-1alpha, CCL27, IP-10, CCR4, CCR5, CXCR3, PD1, PDL2, IL-3, IFN-beta, TIM1, and TLR2 but low antigen-specific cellular responses. J Infect Dis 198:1514–1519

    Article  PubMed  PubMed Central  Google Scholar 

  42. Copeland RA (2000) Enzymes: a practical introduction to structure, mechanism, and data analysis, 2nd edn. Wiley-VCH, New York

  43. Matfield M, Badawi R, Brammar WJ (1985) Rec-dependent and Rec-independent recombination of plasmid-borne duplications in Escherichia coli K12. Mol Gen Genet 199:518–523

    Article  CAS  PubMed  Google Scholar 

  44. Seed B (1983) Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo. Nucleic Acids Res 11:2427–2445

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Watt VM, Ingles CJ, Urdea MS et al (1985) Homology requirements for recombination in Escherichia coli. Proc Natl Acad Sci USA 82:4768–4772

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ninomiya Y, Suzuki K, Ishii C et al (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci USA 101:12248–12253

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  47. Zhang X, Chen W, Zhang Y et al (2012) Deletion of ku homologs increases gene targeting frequency in Streptomyces avermitilis. J Ind Microbiol Biotechnol 39:917–925

    Article  CAS  PubMed  Google Scholar 

  48. Shuman S, Glickman MS (2007) Bacterial DNA repair by non-homologous end joining. Nat Rev Microbiol 5:852–861

    Article  CAS  PubMed  Google Scholar 

  49. Pardo B, Gomez-Gonzalez B, Aguilera A (2009) DNA repair in mammalian cells: DNA double-strand break repair: how to fix a broken relationship. Cell Mol Life Sci 66:1039–1056

    Article  CAS  PubMed  Google Scholar 

  50. Mahaney BL, Meek K, Lees-Miller SP (2009) Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. Biochem J 417:639–650

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Liang F, Romanienko PJ, Weaver DT et al (1996) Chromosomal double-strand break repair in Ku80-deficient cells. Proc Natl Acad Sci USA 93:8929–8933

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  52. Moore JK, Haber JE (1996) Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol 162:164–2173

    Google Scholar 

  53. Wilson TE, Topper LM, Palmbos PL (2003) Non-homologous end-joining: bacteria join the chromosome breakdance. Trends Biochem Sci 28:62–66

    Article  CAS  PubMed  Google Scholar 

  54. Ishibashi K, Suzuki K, Ando Y et al (2006) Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora. Proc Natl Acad Sci USA 103:14871–14876

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  55. Nishizawa-Yokoi A, Nonaka S, Saika H et al (2012) Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice. New Phytol 196:1048–1059

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Chayot R, Montagne B, Mazel D et al (2010) An end-joining repair mechanism in Escherichia coli. Proc Natl Acad Sci USA 107:2141–2146

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  57. Ikeda H, Moriya K, Matsumoto T (1980) In vitro study of illegitimate recombination: involvement of DNA gyrase. Cold Spring Harb Symp Quant Biol 45:399–408

    Article  Google Scholar 

  58. Ikeda H, Aoki K, Naito A (1982) Illegitimate recombination mediated in vitro by DNA gyrase of Escherichia coli: structure of recombinant DNA molecules. Proc Natl Acad Sci USA 79:3724–3728

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  59. Shimizu H, Yamaguchi H, Ikeda H (1995) Molecular analysis of lbio transducing phage produced by oxolinic acid-induced illegitimate recombination in vivo. Genetics 140:889–896

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Ashizawa Y, Yokochi T, Ogata Y et al (1999) Mechanism of DNA gyrase-mediated illegitimate recombination: characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype. J Mol Biol 289:447–458

    Article  CAS  PubMed  Google Scholar 

  61. Nöllmann M, Crisona NJ, Arimondo PB (2007) Thirty years of Escherichia coli DNA gyrase: from in vivo function to single-molecule mechanism. Biochimie 89:490–499

    Article  PubMed  Google Scholar 

  62. Mateos-Gomez PA, Gong F, Nair N et al (2015) Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination. Nature 518:254–257

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  63. Ceccaldi R, Liu JC, Amunugama R et al (2015) Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair. Nature 518:258–262

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  64. Gupta R, Barkan D, Redelman-Sidi G et al (2011) Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways. Mol Microbiol 79:316–330

    Article  CAS  PubMed  Google Scholar 

  65. Heaton BE, Barkan D, Bongiorno P et al (2014) Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection. Infect Immun 82:3177–3185

    Article  PubMed  PubMed Central  Google Scholar 

  66. Branzei D, Foiani M (2008) Regulation of DNA repair throughout the cell cycle. Nature Rev Mol Cell Biol 9:297–308

    Article  CAS  Google Scholar 

  67. Cox MM (1991) The RecA protein as a recombinational repair system. Mol Microbiol 5:1295–1299

    Article  CAS  PubMed  Google Scholar 

  68. Kelley JA, Knight KL (1997) Allosteric regulation of RecA protein function is mediated by Gln194. J Chem Biol 272:25778–25782

    Article  CAS  Google Scholar 

  69. Voloshin ON, Wang L, Camerini-Otero RD (2000) The homologous pairing domain of RecA also mediates the allosteric regulation of DNA binding and ATP hydrolysis: a remarkable concentration of functional residues. J Mol Biol 303:709–720

    Article  CAS  PubMed  Google Scholar 

  70. De Zutter JK, Forget AL, Logan KM et al (2001) Phe217 regulates the transfer of allosteric information across the subunit interface of the RecA protein filament. Structure 9:47–55

    Article  Google Scholar 

  71. Stasiak A, Egelman EH (1994) Structure and function of RecA-DNA complexes. Experientia 50:192–203

    Article  CAS  PubMed  Google Scholar 

  72. Lovett ST, Drapkin PT, Sutera VA Jr et al (1993) A sister-strand exchange mechanism for recA-independent deletion of repeated DNA sequences in Escherichia coli. Genetics 135:631–642

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Zhou R, Kozlov AG, Roy R et al (2011) SSB functions as a sliding platform that migrates on DNA via reptation. Cell 146:222–232

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Satta G, Gudas LJ, Pardee AB (1979) Degradation of Escherichia coli DNA: evidence for limitation in vivo by protein X, the recA gene product. Mol Gen Genet 168:69–80

    Article  CAS  PubMed  Google Scholar 

  75. Kouzminova EA, Kuzminov A (2004) Chromosomal fragmentation in dUTPase-deficient mutants of Escherichia coli and its recombinational repair. Mol Microbiol 51:1279–1295

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Natural Science Foundation of Henan Province (112300410115); the Natural Science Key Foundation of Department of Education (Henan Province) [13A180483]; and the Program for Innovative Research Team from Henan Province, China (15IRTSTHN014).

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Authors and Affiliations

  1. College of Life Sciences, Henan Agricultural University, Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, Zhengzhou, 450002, China

    Ran Chai, Fang Tian, Huiru Li, Qianlong Yang, Andong Song & Liyou Qiu

  2. School of Life Science and Technology, Henan Institute of Science and Technology, Xinxiang, 453003, China

    Chaohui Zhang

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  1. Ran Chai
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Chai, R., Zhang, C., Tian, F. et al. Recombination function and recombination kinetics of Escherichia coli single-stranded DNA-binding protein. Sci. Bull. 61, 1594–1604 (2016). https://doi.org/10.1007/s11434-016-1160-5

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