Isolation and characterization of replication protein A (RP‐A) from tobacco cells

FEBS Letters

Buck, Kenneth W¹  Garcia-Maya, Mitla M¹ 

[1] Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2BB, UK

关键词: Tobacco;    Replication protein A;    DNA polymerase;    Single-stranded DNA-binding protein;    BSA;    bovine serum albumin;    DNase;    deoxyribonuclease;    ds;    double-stranded;    DTT;    dithiothreitol;    EDTA;    ethylenediaminetetraacetic acid;    EGTA;    ethylene glycol-bis(β-aminoethyl ether) N;    N;    N′;    N′-tetraacetic acid;    RP-A;    replication protein A;    ss;    single-stranded;    SSB;    ssDNA-binding protein;    HEPES;    N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid];    MES;    2-[morpholino]ethanesulphonic acid;    PMSF;    phenylmethylsulphonyl fluoride;    SDS-PAGE;    sodium dodecyl sulphate–polyacrylamide gel electrophoresis;    TCA;    trichloroacetic acid;    Tris;    tris[hydroxymethyl]aminomethane;   

DOI  :  10.1016/S0014-5793(97)00897-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown