The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. In this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast's URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutLE36K ). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G < C:C < G:A < C:A, A:A, G:G, T:T, T:G, A:C, C:T < G:T, T:C as the one prevalent in Pseudomonas putida. This information is vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.

中文翻译：

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通过pyrF基因的诱变ssDNA重组揭示恶臭假单胞菌的错配修复体系。

错配修复（MMR）系统是原核细胞用于确保DNA复制保真度的关键分子装置之一。大肠杆菌的典型MMR涉及3种蛋白（由mutS，mutL和mutH编码），土壤细菌假单胞菌恶臭假单胞菌只有2个真正的同源物（mutS和mutL），并且该简化系统对不同类型错配的敏感性为未知。在这种背景下，通过对pyrF基因（相当于酵母URA3的原核生物）进行单链（ss）DNA重组与诱变性寡核苷酸（代表在任何一种野生型条件下可能发生的任何配对错误）的永久性删除，来检查该细菌对MMR的敏感性。 mutS或mutL活性的暂时丧失（由热诱导型显性负等位基因mutLE36K引起）。对含氟乳清酸（5FOA，野生型PyrF的靶标）有抗性的克隆所携带的单核苷酸突变的分析指出了禁止和耐受的单核苷酸替代，并揭示了错配识别的明确等级。结果数据明确地建立了层次结构A：G <C：C <G：A <C：A，A：A，G：G，T：T，T：G，A：C，C：T <G：T ，T：C是恶臭假单胞菌中普遍存在的一种。该信息对于实现针对该生物技术重要物种的单核苷酸变化的重组策略至关重要。A <C：A，A：A，G：G，T：T，T：G，A：C，C：T <G：T，T：C是恶臭假单胞菌中常见的一种。该信息对于实现针对该生物技术重要物种的单核苷酸变化的重组策略至关重要。A <C：A，A：A，G：G，T：T，T：G，A：C，C：T <G：T，T：C是恶臭假单胞菌中常见的一种。该信息对于实现针对该生物技术重要物种的单核苷酸变化的重组策略至关重要。