Dissection of bacteriophage λ site-specific recombination using synthetic peptide combinatorial libraries
Dissection of bacteriophage λ site-specific recombination using synthetic peptide combinatorial libraries
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DOI：
10.1006/jmbi.2000.3828
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发表时间：
2000-06-23
影响因子：
5.6
通讯作者：
Segall, A
中科院分区：
文献类型：
--
作者：
Cassell, G;Klemm, M;Segall, A
各种各样的工具已被用来剖析生化途径，抑制剂是其中的主要。组合方法使得寻找抑制剂的效率大大提高。我们已经应用这样的方法来识别六肽，其抑制由噬菌体λ整合酶蛋白介导的位点特异性重组反应中的不同步骤。整合酶的机制仍然不完全清楚，在很大程度上是因为几个途径中间体仍然难以分离。整合酶催化的重组非常有效，但如果被阻断，它对底物是高度可逆的;这种组合使得一些中间体非常短暂。我们已经使用合成肽组合库来筛选在不同阶段影响重组途径的六肽，并且已经确定了两个肽家族：一个可能阻断DNA切割，另一个可能稳定霍利迪连接中间体。这些肽不类似于整合酶的部分或途径中的任何其他辅助功能。基于酶促反应的抑制以及反应中间体的积累的六肽文库的去卷积是找到用于剖析生化途径的有用工具的新方法。(C)北京大学出版社.
A wide variety of tools have been used to dissect biochemical pathways, inhibitors being chief among them. Combinatorial approaches have made the search for inhibitors much more efficient. We have applied such an approach to identify hexapeptides which inhibit different steps in a site-specific recombination reaction mediated by the bacteriophage lambda integrase protein. Integrase's mechanism is still incompletely understood, in large part because several pathway intermediates remain hard to isolate. Integrase-catalyzed recombination is very efficient, but if blocked, it is highly reversible to substrates; this combination makes some intermediates exceedingly transient. We have used synthetic peptide combinatorial libraries to screen for hexapeptides that affect the recombination pathway at different stages, and have identified two families of peptides: one probably blocks DNA cleavage, the other may stabilize the Holliday junction intermediates. These peptides do not resemble parts of integrase or any of the other helper functions in the pathway. The deconvolution of hexapeptide libraries based both on inhibition of an enzymatic reaction as well as on accumulation of reaction intermediates is a novel approach to finding useful tools for dissecting a biochemical pathway. (C) 2000 Academic Press.