title: Kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab) coat protein and its mutants with ssDNA.
abstract: Gemini viral assembly and transport of viral DNA into nucleus for replication, essentially involve DNA-coat protein interactions. The kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali recombinant coat protein (rCP) with DNA was studied by electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR). The rCP interacted with ssDNA with a K(A), of 2.6+/-0.29 x 10(8) M(-1) in a sequence non-specific manner. The CP has a conserved C2H2 type zinc finger motif composed of residues C68, C72, H81 and H85. Mutation of these residues to alanine resulted in reduced binding to DNA probes. The H85A mutant rCP showed the least binding with approximately 756 fold loss in the association rate and a three order magnitude decrease in the binding affinity as compared to rCP. The CP-DNA interactions via the zinc finger motif could play a crucial role in virus assembly and in nuclear transport.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Poornima C G
authors.initials: PC
authors.lastname: Priyadarshini
authors.forename: H S
authors.initials: HS
authors.lastname: Savithri
mesh_terms: D000595:Amino Acid Sequence; D053839:Begomovirus / Q000235:genetics* / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015342:DNA Probes; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D015342:DNA Probes; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins