title: Isolation and characterization of replication protein A (RP-A) from tobacco cells.
abstract: Replication protein A (RP-A) was isolated from tobacco suspension cells and purified to near homogeneity by a procedure involving isolation of protoplasts, preparation of nuclei, nuclear lysis, binding to a column of single-stranded (ss) DNA cellulose and elution at different salt concentrations. The purified protein contained three subunits with molecular masses of 70, 34 and 14 kDa, and was free from nuclease activity. Tobacco RP-A had a high affinity for ssDNA. Binding competition experiments indicated only a weak affinity for double-stranded DNA and no detectable affinity for ssRNA. Photochemical cross-linking experiments indicated that the 70 kDa subunit has the ssDNA-binding activity. Tobacco RP-A was able to stimulate the activity of a tobacco alpha-like DNA polymerase about 4-fold. This is the first isolation of RP-A from a plant and the procedure may be generally applicable to other plant species.
authors.affiliation: Department of Biology, Imperial College of Science, Technology and Medicine, London, UK.
authors.forename: M M
authors.initials: MM
authors.lastname: Garcia-Maya
authors.forename: K W
authors.initials: KW
authors.lastname: Buck
mesh_terms: D002478:Cells, Cultured; D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D004259:DNA-Directed DNA Polymerase / Q000187:drug effects / Q000378:metabolism; D010947:Plants, Toxic; D051716:Replication Protein A; D014026:Nicotiana / Q000737:chemistry*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D051716:Replication Protein A; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases