title: R-loop induced stress response by second (p)ppGpp synthetase in Mycobacterium smegmatis: functional and domain interdependence.
abstract: Persistent R-loops lead to replicative stress due to RNA polymerase stalling and DNA damage. RNase H enzymes facilitate the organisms to survive in the hostile condition by removing these R-loops. MS_RHII-RSD was previously identified to be the second (p)ppGpp synthetase in Mycobacterium smegmatis. The unique presence of an additional RNase HII domain raises an important question regarding the significance of this bifunctional protein. In this report, we demonstrate its ability to hydrolyze R-loops in Escherichia coli exposed to UV stress. MS_RHII-RSD gene expression was upregulated under UV stress, and this gene deleted strain showed increased R-loop accumulation as compared to the wild type. The domains in isolation are known to be inactive, and the full length protein is required for its function. Domain interdependence studies using active site mutants reveal the necessity of a hexamer form with high alpha helical content. In previous studies, bacterial RNase type HI has been mainly implicated in R-loop hydrolysis, but in this study, the RNase HII domain containing protein showed the activity. The prospective of this differential RNase HII activity is discussed. This is the first report to implicate a (p)ppGpp synthetase protein in R-loop-induced stress response.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, India.
authors.forename: Sushma
authors.initials: S
authors.lastname: Krishnan
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, India.
authors.forename: Anushya
authors.initials: A
authors.lastname: Petchiappan
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B152TT, UK.
authors.forename: Albel
authors.initials: A
authors.lastname: Singh
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B152TT, UK.
authors.forename: Apoorva
authors.initials: A
authors.lastname: Bhatt
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, India. dipankar@mbu.iisc.ernet.in.
authors.forename: Dipankar
authors.initials: D
authors.lastname: Chatterji
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D020134:Catalytic Domain; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D006868:Hydrolysis; D008025:Ligases / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism; D013312:Stress, Physiological / Q000502:physiology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D012321:DNA-Directed RNA Polymerases; C091139:ribonuclease HII; D016914:Ribonuclease H; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases