title: Mutational analysis of the helicase domain of a replication initiator protein reveals critical roles of Lys 272 of the B' motif and Lys 289 of the β-hairpin loop in geminivirus replication.
abstract: Replication initiator protein (Rep) is indispensable for rolling-circle replication of geminiviruses, a group of plant-infecting circular ssDNA viruses. However, the mechanism of DNA unwinding by circular ssDNA virus-encoded helicases is unknown. To understand geminivirus Rep function, we compared the sequence and secondary structure of Rep with those of bovine papillomavirus E1 and employed charged residue-to-alanine scanning mutagenesis to generate a set of single-substitution mutants in Walker A (K227), in Walker B (D261, 262), and within or adjacent to the B' motif (K272, K286 and K289). All mutants were asymptomatic and viral accumulation could not be detected by Southern blotting in both tomato and N. benthamiana plants. Furthermore, the K272 and K289 mutants were deficient in DNA binding and unwinding. Biochemical studies and modelling data based on comparisons with the known structures of SF3 helicases suggest that the conserved lysine (K289) located in a predicted β-hairpin loop may interact with ssDNA, while lysine 272 in the B' motif (K272) located on the outer surface of the protein is presumably involved in coupling ATP-induced conformational changes to DNA binding. To the best of our knowledge, this is the first time that the roles of the B' motif and the adjacent β-hairpin loop in geminivirus replication have been elucidated.
authors.affiliation: Department of Biotechnology, Jamia Hamdard University, New Delhi, India.|School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Biju
authors.initials: B
authors.lastname: George
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Rajrani
authors.initials: R
authors.lastname: Ruhel
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Mohit
authors.initials: M
authors.lastname: Mazumder
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Veerendra Kumar
authors.initials: VK
authors.lastname: Sharma
authors.affiliation: Department of Biotechnology, Jamia Hamdard University, New Delhi, India.
authors.forename: Swatantra Kumar
authors.initials: SK
authors.lastname: Jain
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Samudrala
authors.initials: S
authors.lastname: Gourinath
authors.affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Supriya
authors.initials: S
authors.lastname: Chakraborty
mesh_terms: D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D017791:Geminiviridae / Q000201:enzymology* / Q000302:isolation & purification / Q000502:physiology*; D018551:Solanum lycopersicum / Q000821:virology; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010935:Plant Diseases / Q000821:virology; D017433:Protein Structure, Secondary; D014026:Nicotiana / Q000821:virology; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D050505:Mutant Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases