Bhosale Ankita, Sen Ranjan
Laboratory of Transcription, Center for DNA Fingerprinting and Diagnostics, Uppal, Hyderabad 500039, India.
Graduate Studies, Manipal University, Manipal, Karnataka.
Biochem J. 2025 May 26;482(11):BCJ20253089. doi: 10.1042/BCJ20253089.
Ribonuclease HI (rnhA) removes the deleterious RNA:DNA hybrids (RDHs) by cleaving its RNA component. The bacterial transcription terminator Rho is an RNA-dependent 5' → 3' helicase capable of unwinding RDH formed on a single-stranded RNA in vitro. We hypothesize that Rho might be directly involved in RDH removal in vivo. Here, we demonstrate that Rho primary RNA-binding site (PBS) mutants defective in RNA binding and helicase activity are synthetically lethal specifically when RNase HI is absent. This lethality was not observed in the absence of RNase HII (rnhB) alone. Rho-PBS mutants in an rnhA- strain exhibited increased plasmid-concatemer and plasmid copy number, altered cell morphology, and were highly susceptible to DNA-damaging agents. These Rho mutants increased the accumulation of RDHs in vivo, suggesting defects in the RDH removal process. Rho was colocalized to RDHs in vivo when RNase HI was absent. Certain catalytically inactive mutants of RNase H that bind to the RDH blocked the entry of Rho to the RDH, inducing cell death, indicating the role of Rho in the removal of deleterious RDHs in the absence of RNase HI. Under in vitro conditions, Rho was capable of binding to the RDHs and unwinding them in a rut-site-dependent manner. Therefore, we concluded that in the absence of RNase HI, Rho, by its RNA-dependent helicase activity, is capable of unwinding RDHs in a rut-site-dependent manner. These results establish the non-transcription terminator role of Rho and its functional synergy with RNase HI in vivo.
核糖核酸酶HI（rnhA）通过切割其RNA成分来去除有害的RNA:DNA杂交体（RDHs）。细菌转录终止因子Rho是一种依赖RNA的5'→3'解旋酶，能够在体外解开单链RNA上形成的RDH。我们推测Rho可能在体内直接参与RDH的去除。在这里，我们证明了在RNA结合和解旋酶活性方面存在缺陷的Rho主要RNA结合位点（PBS）突变体，特别是在没有核糖核酸酶HI时是合成致死的。单独缺乏核糖核酸酶HII（rnhB）时未观察到这种致死性。rnhA-菌株中的Rho-PBS突变体表现出质粒串联体增加和质粒拷贝数改变，细胞形态改变，并且对DNA损伤剂高度敏感。这些Rho突变体增加了体内RDH的积累，表明在RDH去除过程中存在缺陷。当没有核糖核酸酶HI时，Rho在体内与RDH共定位。某些与RDH结合的核糖核酸酶H催化无活性突变体阻止了Rho进入RDH，导致细胞死亡，这表明在没有核糖核酸酶HI的情况下，Rho在去除有害RDH中发挥作用。在体外条件下，Rho能够结合到RDH上并以依赖rut位点的方式解开它们。因此，我们得出结论，在没有核糖核酸酶HI的情况下，Rho通过其依赖RNA的解旋酶活性，能够以依赖rut位点的方式解开RDH。这些结果确立了Rho的非转录终止因子作用及其在体内与核糖核酸酶HI的功能协同作用。
