Wittung P, Funk M, Jernström B, Nordén B, Takahashi M
Department of Physical Chemistry, Chalmers University of Technology, Gothenburg, Sweden.
FEBS Lett. 1995 Jul 10;368(1):64-8. doi: 10.1016/0014-5793(95)00600-e.
A technique has been developed to probe directly RecA-DNA interactions by the use of the fluorescent chromophore, (+)anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), covalently attached to DNA. The 24-mer oligonucleotide 5'-d(CTACTAAACATGTACAAATCATCC) was specifically modified on the exocyclic nitrogen of the central guanine, to yield a trans-adduct. Upon interaction of the modified oligonucleotide with RecA we find an increase in BPDE fluorescence and a rather high fluorescence anisotropy, suggesting a restricted motion of the BPDE-oligonucleotide in the protein filament. In the presence of the cofactor ATP gamma S, binding of two oligonucleotides, identical or complementary in sequence, in the RecA filament is possible. The RecA-DNA complex is, however, more stable when the sequences are complementary; in addition, a shift in the BPDE emission peaks is observed. In the presence of ATP (and an ATP regeneration system), the RecA-DNA interaction between two complementary oligonucleotides is changes, and we now find protein-mediated renaturation to occur.
已开发出一种技术，通过使用共价连接到DNA上的荧光发色团（+）反式苯并[a]芘-7,8-二氢二醇-9,10-环氧化物（BPDE）来直接探测RecA与DNA的相互作用。24聚体寡核苷酸5'-d（CTACTAAACATGTACAAATCATCC）在中央鸟嘌呤的环外氮上进行了特异性修饰，以产生反式加合物。当修饰的寡核苷酸与RecA相互作用时，我们发现BPDE荧光增加且荧光 anisotropy 相当高，这表明BPDE-寡核苷酸在蛋白质丝中的运动受到限制。在辅因子ATPγS存在的情况下，有可能在RecA丝中结合两个序列相同或互补的寡核苷酸。然而，当序列互补时，RecA-DNA复合物更稳定；此外，观察到BPDE发射峰发生了位移。在ATP（和ATP再生系统）存在的情况下，两个互补寡核苷酸之间的RecA-DNA相互作用发生变化，我们现在发现发生了蛋白质介导的复性。
