CHARACTERIZATION OF THE DNA-BINDING ACTIVITY OF STABLE RECA-DNA COMPLEXES - INTERACTION BETWEEN THE 2 DNA-BINDING SITES WITHIN RECA HELICAL FILAMENTS
CHARACTERIZATION OF THE DNA-BINDING ACTIVITY OF STABLE RECA-DNA COMPLEXES - INTERACTION BETWEEN THE 2 DNA-BINDING SITES WITHIN RECA HELICAL FILAMENTS
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DOI：
10.1016/0022-2836(90)90307-8
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发表时间：
1990-03-05
影响因子：
5.6
通讯作者：
STASIAK, A
中科院分区：
文献类型：
--
作者：
MULLER, B;KOLLER, T;STASIAK, A
描述了由ATP γ S（ATP的缓慢水解类似物）稳定的纯化RecA-DNA复合物的DNA结合、退火和重组活性。电泳分析，DNA酶保护实验和电子显微镜观察表明，饱和的RecA复合物形成的单链或双链DNA能够容纳一个额外的单链DNA与化学计量的约一个核苷酸的添加的单链DNA每个核苷酸或碱基对，分别在复杂的DNA居民。这种链摄取不依赖于所添加的DNA分子和驻留的DNA分子之间的互补性或同源性。在复合物中，进入的和驻留的单链DNA分子非常接近，因为两条链可以在它们互补的情况下退火。当添加的DNA与复合链同源时，与单链DNA形成的稳定RecA复合物可有效结合双链DNA，然后引发伴侣DNA分子之间的链交换反应。与同源双链DNA相关的RecA-单链DNA复合物的电子显微镜表明，双链DNA的一部分被吸收到复合物中，并与常驻单链配准。我们的实验表明，RecA螺旋丝内的DNA结合位点可以被单链或双链DNA占据。据推测，相同的第一个DNA结合位点被RecA在其对单链或双链DNA的聚合过程中使用，并且第二个DNA结合位点可用于随后的蛋白质饱和复合物与裸DNA的相互作用。额外的DNA被纳入RecA-DNA复合物的方式显示出合作的特点，它有助于解释如何避免拓扑问题，在RecA介导的同源重组。
The DNA-binding, annealing, and recombinational activities of purified RecA-DNA complexes stabilized by ATP.gamma.S (a slowly hydrolysable analog of ATP) are described. Electrophoretic analysis, DNase protection experiments and observations by electron microscopy suggest that saturated RecA complexes formed with single- or double-stranded DNA are able to accommodate an additional single strand of DNA with a stoichiometry of about one nucleotide of added single-stranded DNA per nucleotide or base-pair, respectively, of DNA resident in the complex. This strand uptake is independent of complementarity or homology between the added and resident DNA molecules. In the complex, the incoming and resident single-stranded DNA molecules are in close proximity as the two strands can anneal in case of their complementarity. Stable RecA complexes formed with single-stranded DNA bind double-stranded DNA efficiently when the added DNA is homologous to the complexed strand and then initiate a strand exchange reaction between the partner DNA molecules. Electron microscopy of the RecA-single-stranded DNA complexes associated with homologous double-stranded DNA suggests that a portion of duplex DNA is take into the complex and placed in register with the resident single strand. Our experiments indicate that both DNA binding sites within RecA helical filaments can be occupied by either single- or double-stranded DNA. Presumably, the same first DNA binding site is used by RecA during its polymerization on single- or double-stranded DNA and the second DNA binding site becomes available for subsequent interaction of the protein-saturated complexes with naked DNA. The way by which additional DNA is taken into RecA-DNA complexes shows co-operative character and its helps to explain how topological problems are avoided during RecA-mediated homologous recombination.