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 ne...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.展开更多
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)
文摘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.
