Interaction of different oligomeric states of hexameric DNA‐helicase RepA with single‐stranded DNA studied by analytical ultracentrifugation

FEBS Letters

Holzwarth, Josef F.²  Behlke, Joachim³  Frank, Joachim²  Xu, Hai¹  Saenger, Wolfram¹ 

[1] Institut für Kristallographie, Freie Universität Berlin, Takustr. 6, D-14195 Berlin, Germany;Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4–6, D-14195 Berlin, Germany;Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, D-13122 Berlin, Germany

关键词: RepA;    Single-stranded DNA;    Equilibrium binding;    Stoichiometry;    Analytical ultracentrifugation;    ssDNA;    single-stranded DNA;    dsDNA;    double-stranded DNA;    ATPγS;    adenosine-5′-O-(3-thiotriphosphate);    MES;    2-(N-morpholino)ethane sulfonic acid;    (RepA)6;    RepA hexamer;   

DOI  :  10.1016/S0014-5793(00)02026-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA-helicase RepA at pH 5.8 and 7.6. At pH 7.6, a molecular mass of 179.5±2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA)₆. At pH 5.8, (RepA)₆ associates to form a dimer with a molecular mass of 366.2±4.1 kDa. Analytical ultracentrifugation was also applied to characterize the interaction of single-stranded DNA (ssDNA) with the two different oligomeric states of (RepA)₆ at pH 5.8 and 7.6. The dissociation constants, K _d, for the equilibrium binding of (dA)₃₀ to the (RepA)₆ dimer at pH 5.8 and to (RepA)₆ at pH 7.6 were determined at 10°C in the presence of 0.5 mM ATPγS, 10 mM MgCl₂ and 60 mM NaCl as K _d5.8=0.94±0.13 μM at pH 5.8 and K _d7.6=25.4±6.4 μM at pH 7.6. The stoichiometries, n, for the two complexes (dA)₃₀/(RepA)₆ dimer and (dA)₃₀/(RepA)₆ at pH 5.8 and 7.6 were calculated from the corresponding binding curves. At pH 5.8 one (dA)₃₀ molecule was bound per (RepA)₆ dimer, while at pH 7.6 one (dA)₃₀ molecule was bound to one (RepA)₆. Binding curves were compatible with a single ssDNA binding site present on the (RepA)₆ dimer and on (RepA)₆, respectively, with no indication of cooperativity. (RepA)₆ tends to form larger aggregates under acidic conditions (pH<6.0) which are optimal for ssDNA binding. In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA)₆ dimer was observed both in the absence and presence of (dA)₃₀.

【 授权许可】

Unknown