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首页> 外文期刊>Journal of Molecular Biology >The lambda Gam protein inhibits RecBCD binding to dsDNA ends
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The lambda Gam protein inhibits RecBCD binding to dsDNA ends

机译：λGam蛋白抑制RecBCD与dsDNA末端的结合

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Inactivation of the Escherichia coli RecBCD enzyme by the lambda Gam protein is an essential step that accompanies the X Red proteins for gene replacement using recombineering technology. It has been shown that Gam inhibits all the activities of RecBCD to the same extent. Nonetheless, some in vivo properties of recBCD mutants cannot be mimicked effectively by the expression of gam in vivo. An examination of the mechanism of Gam's inhibition of RecBCD was performed, and it was found that Gam inhibits the binding of RecBCD to double-stranded DNA ends, even if RecBCD is bound to DNA before its interaction with Gam. When ATP is added to the reaction to induce helicase activity; most of the reaction is inhibited by Gam, but residual amounts of unwinding are detected, despite a 40-fold excess of Gam/RecBCD. The same inhibitory effect of Gam was seen on RecBCD that had been modified by the P22 anti-RecBCD protein Abc2, though the inhibitory effect was diminished due to the tighter binding of Abc2-modified RecBCD to double-stranded DNA ends. These data suggest that cells containing Gam-expressing plasmids retain a small amount of uninhibited enzyme. Given the suspected. instability of Gam in vivo, care must be taken when interpreting results from experiments containing Gam-inhibited RecBCD species. A revised model is proposed for Gam-induced radioresistance of E. coli to ionizing radiation. (c) 2007 Elsevier Ltd. All rights reserved.
机译：λGam蛋白使大肠杆菌RecBCD酶失活是X Red蛋白质伴随重组过程中基因置换所必不可少的步骤。已经表明，Gam在相同程度上抑制了RecBCD的所有活性。但是，recBCD突变体的某些体内特性不能通过gam在体内的表达有效地模仿。进行了Gam抑制RecBCD的机制的研究，发现即使RecBCD在与Gam相互作用之前已经结合到DNA上，Gam也抑制了RecBCD与双链DNA末端的结合。当将ATP添加到反应中以诱导解旋酶活性时;尽管Gam / RecBCD过量40倍，但大多数反应均受Gam抑制，但仍检测到残留的解链量。尽管由于Abc2修饰的RecBCD与双链DNA末端更紧密的结合而使抑制作用减弱，但Gam对RecBCD的抑制作用已被P22抗RecBCD蛋白Abc2修饰。这些数据表明，含有表达Gam的质粒的细胞保留了少量未抑制的酶。鉴于怀疑。 Gam在体内不稳定，在解释含有Gam抑制的RecBCD物种的实验结果时必须小心。提出了针对Gam诱导的大肠杆菌对电离辐射的抗辐射性的修订模型。 （c）2007 Elsevier Ltd.保留所有权利。

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