RecA protein--promoted lambda repressor cleavage: complementation between RecA441 and RecA430 proteins in vitro.
RecA protein--promoted lambda repressor cleavage: complementation between RecA441 and RecA430 proteins in vitro.
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RecA 蛋白——促进 lambda 阻遏蛋白裂解：体外 RecA441 和 RecA430 蛋白之间的互补。
DOI：
10.1007/bf00328696
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Roberts,JW
中科院分区：
文献类型：
--
作者：
Moreau,PL;Roberts,JW
在腺嘌呤存在的情况下，转移至42° C后，在recA 441突变溶原细胞中发生λ原噬菌体的诱导。如果RecA 441蛋白的合成通过recA 441基因中的第二个突变recA 453的存在而维持在低基础水平，则前噬菌体λ的诱导被阻止。通过在转导噬菌体上引入携带recA 430突变的第二个recA基因，可以恢复诱导前噬菌体λ的能力; RecA 430蛋白本身对λ阻遏物没有活性（Rebollo et al. 1984）。为了解释RecA 430蛋白如何与RecA 441蛋白互补以在arecA 453 -441（recA 430）二倍体溶原细胞中提供λ阻遏物切割，我们在体外表征了由这些蛋白的混合物催化的切割反应。我们的结果表明，在dATP存在下，RecA 441和RecA 430蛋白在单链DNA上形成混合多聚体，其中RecA 441蛋白分子增强RecA 430蛋白分子的DNA结合亲和力，而RecA 430蛋白分子不支持λ阻遏物的切割。我们发现SSB蛋白不能替代RecA 430蛋白恢复arecA 453 - 441溶原细胞中λ阻遏物的切割。比较长单链DNA和（dA）14寡核苷酸对RecA 441蛋白在不同三磷酸核苷（NTP）存在下定向切割λ阻遏物的刺激作用表明，RecA 441蛋白与单链DNA的协同结合稳定了RecA蛋白-DNA复合物，使它们保持完整足够长的时间以支持λ阻遏物的切割。我们的结论是，在arecA 453 - 441细胞中RecA 441蛋白的低基础水平足以切割λ阻遏物，在正常基础水平的RecA 430蛋白也存在的条件下，允许在通常存在于细胞中的单链DNA区域上形成混合多聚体。
Induction of prophage λ occurs inrecA441mutant lysogens after a shift to 42° C in the presence of adenine. If the synthesis of RecA441 protein is maintained at a low basal level by the presence of a second mutation in therecA441gene,recA453, induction of prophage λ is prevented. The ability to induce prophage λ is restored by the introduction, on a transducing phage, of a secondrecAgene carrying therecA430mutation; by itself, the RecA430 protein is devoid of activity against the λ repressor (Rebollo et al. 1984). In order to explain how the RecA430 protein might complement the RecA441 protein to provide λ repressor cleavage in arecA453-441 (recA430)diploid lysogen, we characterized the cleavage reaction catalysed by a mixture of these proteins in vitro. Our results suggest that, in the presence of dATP, the RecA441 and RecA430 proteins form mixed multimers on single-stranded DNA, in which the RecA441 protein molecules enhance the DNA binding affinity of RecA430 protein molecules, but RecA430 protein molecules support no cleavage of the λ repressor.Although the effects of the RecA430 and single-strand binding (SSB) proteins are similar in vitro, we show that the SSB protein cannot substitute for the RecA430 protein in restoring λ repressor cleavage in arecA453-441lysogen. Comparison of the stimulatory effect of long single-stranded DNA with that of (dA)14oligonucleotides on the RecA441 protein-directed cleavage of the λ repressor in the presence of various nucleoside triphosphates (NTPs) indicates that the cooperative binding of the RecA441 protein to single-stranded DNA stabilizes the RecA protein-DNA complexes so that they remain intact long enough to support cleavage of the λ repressor. We conclude that the low basal level of the RecA441 protein in arecA453-441cell is sufficient to cleave the λ repressor, under conditions where a normal basal level of RecA430 protein is also present allowing the formation of mixed multimers on single-stranded DNA regions normally present in the cell.