Cyanobacterial Stress Biology and Biotechnology Section, Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India.
Radiaiton Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India; Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400094, India.
Gene. 2021 Jul 1;788:145663. doi: 10.1016/j.gene.2021.145663. Epub 2021 Apr 20.
The high radioresistance of Nostoc sp. strain PCC7120 is indicative of a robust DNA repair pathway. In the absence of NHEJ pathway and the canonical RecBCD proteins, the RecF pathway proteins are expected to play an important role in double strand break repair in this organism. The RecF, RecO and RecR proteins which are central to the RecF pathway have not been characterised in the ancient cyanobacteria, several of which are known to be radioresistant. The characterisation of these proteins was initiated through a mix of in silico, expression and complementation analysis. Differential expression of the recF, recO and recR genes was observed both at the transcript and the protein level under normal growth condition, which did not change significantly upon exposure to DNA damage stresses. Expression of RecR as a 23 kDa protein in vivo in Nostoc PCC7120 confirmed the re-annotation of the initiation codon of the gene (alr4977) to a rare initiation codon 'GTT' 267 bases upstream of the annotated initiation codon. Of the three proteins, Nostoc RecO and RecR proteins could complement the corresponding mutations in Escherichia coli, but not RecF. The Nostoc RecO protein exhibited low sequence and structural homology with other bacterial RecO protein, and was predicted to have a longer loop region. Phylogenetic as well as sequence analysis revealed high conservation among bacterial RecR proteins and least for RecO. In silico analysis revealed a comparatively smaller interactome for the Nostoc RecF, RecO and RecR proteins compared to other bacteria, with RecO predicted to interact with both RecF and RecR. The information gathered can form a stepping stone to further characterise these proteins in terms of deciphering their interactome, biochemical and physiological activities. This would help in establishing their importance in RecF pathway of DSB repair in Nostoc PCC7120.
念珠藻 PCC7120 的高放射抗性表明其具有强大的 DNA 修复途径。在不存在非同源末端连接途径和典型的 RecBCD 蛋白的情况下，RecF 途径蛋白预计在该生物中二链断裂修复中发挥重要作用。RecF、RecO 和 RecR 蛋白是 RecF 途径的核心，在古老的蓝细菌中尚未得到表征，其中一些已知具有放射抗性。这些蛋白的表征是通过计算机分析、表达和互补分析的混合方法开始的。在正常生长条件下，在转录和蛋白质水平上都观察到 recF、recO 和 recR 基因的差异表达，在暴露于 DNA 损伤应激时没有显著变化。RecR 作为 23 kDa 蛋白在体内表达在 Nostoc PCC7120 中证实了对基因（alr4977）起始密码子的重新注释，该基因的起始密码子为罕见的起始密码子“GTT”，位于注释起始密码子上游 267 个碱基。在这三种蛋白中， Nostoc RecO 和 RecR 蛋白可以互补大肠杆菌中相应的突变，但不能互补 RecF。Nostoc RecO 蛋白与其他细菌的 RecO 蛋白的序列和结构同源性较低，预测具有更长的环区。系统发育和序列分析表明，细菌 RecR 蛋白之间高度保守，而 RecO 蛋白最少保守。计算机分析表明，与其他细菌相比， Nostoc RecF、RecO 和 RecR 蛋白的相互作用组相对较小，预测 RecO 与 RecF 和 RecR 相互作用。收集的信息可以为进一步阐明这些蛋白的相互作用组、生化和生理活性奠定基础。这将有助于确定它们在 Nostoc PCC7120 的 DSB 修复 RecF 途径中的重要性。
