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Isolation and characterization of replication protein A (RP‐A) from tobacco cells
Author(s) -
Garcia-Maya Mitla M,
Buck Kenneth W
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00897-1
Subject(s) - nuclease , lysis , dna , protein subunit , biochemistry , tandem affinity purification , dna replication , affinity chromatography , nicotiana tabacum , protoplast , tobacco mosaic virus , biology , microbiology and biotechnology , chemistry , enzyme , genetics , virus , gene
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 α‐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.