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Recombination function and recombination kinetics of Escherichia coli single-stranded DNA-binding protein 被引量:1

Recombination function and recombination kinetics of Escherichia coli single-stranded DNA-binding protein
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摘要 It is unknown whether the ss DNA-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 Rec A.However,ssb-dependent recombination occurred later and at a lower recombination rate than the rec A-dependent,probably because ssb expression was about two-fold lower than rec A during the E.coli earlier growth stage.The affinity to substrate and the recombination efficiency of SSB was lower than Rec A,whereas SSB enhanced the catalytic efficiency of Rec A.Knocking out both rec A and ssb led to loss of recombination.Our results confirmed that as SSB has the recombination function itself as an allosteric enzyme,rec Aindependent recombination in E.coli should be ssb-dependent.ssb-dependent recombination may be the third DNA double-strand break repair pathway,in addition to rec Adependent recombination and non-homologous end joining. 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 recom- bination. Our results confirmed that as SSB has the recombi- nation 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.
出处 《Science Bulletin》 SCIE EI CAS CSCD 2016年第20期1594-1604,共11页 科学通报(英文版)
基金 supported by the Natural Science Foundation of Henan Province (112300410115) the Natural Science Key Foundation of Department of Education (Henan Province)[13A180483] the Program for Innovative Research Team from Henan Province,China (15IRTSTHN014)
关键词 ss DNA-binding-protein Homologous recombination Non-homologous recombination Recombination kinetics ssDNA-binding-protein Homologousrecombination Non-homologous recombination.Recombination kinetics
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  • 1Michel B, Grompone G, Flore's MJ et al (2004) Multiple path- ways process stalled replication forks. Proc Natl Acad Sci USA 101:12783-12788.
  • 2Rocha EPC, Comet E, Michel B (2005) Comparative and evo- lutionary analysis of the bacterial homologous recombination systems. PLoS Genet l:e15.
  • 3Jasin 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.
  • 4Zhang Y, Muyrers JP, Testa G et al (2000) DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol 18:1314-1317.
  • 5Sharan SK, Thomason LC, Kuznetsov SG et al (2009) Recom- bineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4:206-223.
  • 6Bell 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.
  • 7Chai R, Qiu C, Liu D et al (2013) f3-Glucan synthase gene overexpression and J3-glucans overproduction in Pleurotus ostreatus using promoter swapping. PLoS ONE 8:e61693.
  • 8Slade D, Radman M (2011) Oxidative stress resistance in Deinococcus radiodurans. Microbiol Mol Biol Rev 75:133-191.
  • 9Dillingham MS, Kowalczykowski SC (2008) RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiol Mol Biol Rev 72:642-671.
  • 10Smith 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.

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