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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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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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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DOI: https://doi.org/10.1007/s11434-016-1160-5