Cerutti H., Jagendorf A. T.
Section of Plant Biology, Cornell University, Ithaca, New York 14853.
Plant Physiol. 1993 May;102(1):145-153. doi: 10.1104/pp.102.1.145.
The occurrence of DNA recombination in plastids of higher plants is well documented. However, little is known at the enzymic level. To begin dissecting the biochemical mechanism(s) involved we focused on a key step: strand transfer between homologous parental DNAs. We detected a RecA-like strand transfer activity in stromal extracts from pea (Pisum sativum L.) chloroplasts. Formation of joint molecules requires Mg2+, ATP, and homologous substrates. This activity is inhibited by excess single-stranded DNA (ssDNA), suggesting a necessary stoichiometric relation between enzyme and ssDNA. In a novel assay with Triton X-100-permeabilized chloroplasts, we also detected strand invasion of the endogenous chloroplast DNA by 32P-labeled ssDNA complementary to the 16S rRNA gene. Joint molecules, analyzed by electron microscopy, contained the expected displacement loops.
高等植物质体中DNA重组的发生已有充分记录。然而,在酶学水平上了解甚少。为了开始剖析其中涉及的生化机制,我们聚焦于一个关键步骤:同源亲本DNA之间的链转移。我们在豌豆(Pisum sativum L.)叶绿体的基质提取物中检测到了一种类似RecA的链转移活性。连接分子的形成需要Mg2+、ATP和同源底物。这种活性受到过量单链DNA(ssDNA)的抑制,表明酶与ssDNA之间存在必要的化学计量关系。在一项使用Triton X-100通透化叶绿体的新实验中,我们还检测到与16S rRNA基因互补的32P标记的ssDNA对叶绿体内源DNA的链侵入。通过电子显微镜分析的连接分子包含预期的置换环。