单链DNA结合蛋白SSB/RPA是细菌/真核生物中与ssDNA结合并协调DNA代谢过程如复制的普遍存在和必需的蛋白质。修复,和重组。SSB保护ssDNA免受核酸酶降解,同时还促进/调节参与DNA过程的多种伴侣蛋白的活性。使用Spi-分析,它检测从大肠杆菌染色体中异常切除的λ原蛋白,作为非法重组(IR)发生的量度,我们已经证明SSB在几个DSB切除途径中抑制IR。条件ssb-1突变在非允许温度下产生比recQ失活更高的IR增加。双ssb-1recQ突变体具有更高的IR水平,同时显示减少的同源重组(HR)。值得注意的是,ssb基因过表达补充recQ缺陷抑制IR,表明SSB函数对RecQ是上位性的。过度产生的截短的SSBΔC8蛋白,与ssDNA结合,但不与伴侣蛋白相互作用,只有部分互补的recQ和ssb-1突变,同时导致野生型细菌的IR增加,表明SSB的ssDNA结合是必需的,但不足以有效抑制IR,这需要与RecQ和可能的其他蛋白质相互作用。我们的结果描述了SSB作为大肠杆菌的主要基因组管理员,在抑制IR的同时促进HR。在生理条件下实现高保真DSB修复时,RecQ解旋酶辅助SSB,它控制谁的活动。相反,过量的SSB使得RecQ对于IR抑制是冗余的。
Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi- assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression.