Fukuda Tomoyuki, Nogami Satoru, Ohya Yoshikazu
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bldg. FSB-101, 5-1-5 Kashiwanoha, Kashiwa, Chiba Prefecture, 277-8562 Japan.
Genes Cells. 2003 Jul;8(7):587-602. doi: 10.1046/j.1365-2443.2003.00659.x.
Inteins and group I introns found in prokaryotic and eukaryotic organisms occasionally behave as mobile genetic elements. During meiosis of the yeast Saccharomyces cerevisiae, the site-specific endonuclease encoded by VMA1 intein, VDE, triggers a single double-strand break (DSB) at an inteinless allele, leading to VMA1 intein homing. Besides the accumulating information on the in vitro activity of VDE, very little has been known about the molecular mechanism of intein homing in yeast nucleus.
We developed an assay to detect the product of VMA1 intein homing in yeast genome. We analysed mutant phenotypes of RecA homologs, Rad51p and Dmc1p, and their interacting proteins, Rad54p and Tid1p, and found that they all play critical roles in intein inheritance. The absence of DSB end processing proteins, Sae2p and those in the Mre11-Rad50-Xrs2 complex, also causes partial reduction in homing efficiency. As with meiotic recombination, crossover events are frequently observed during intein homing. We also observed that the absence of premeiotic DNA replication caused by hydroxyurea (HU) or clb5delta clb6delta mutation reduces VDE-mediated DSBs.
The repairing system working in intein homing shares molecular machinery with meiotic recombination induced by Spo11p. Moreover, like Spo11p-induced DNA cleavage, premeiotic DNA replication is a prerequisite for a VDE-induced DSB. VMA1 intein thus utilizes several host factors involved in meiotic and recombinational processes to spread its genetic information and guarantee its progeny through establishment of a parasitic relationship with the organism.
在原核生物和真核生物中发现的内含肽和I组内含子偶尔会表现为可移动遗传元件。在酿酒酵母减数分裂过程中，由VMA1内含肽编码的位点特异性内切酶VDE在无内含肽的等位基因处引发单个双链断裂（DSB），导致VMA1内含肽归巢。除了关于VDE体外活性的信息不断积累外，关于酵母细胞核中内含肽归巢的分子机制知之甚少。
我们开发了一种检测酵母基因组中VMA1内含肽归巢产物的方法。我们分析了RecA同源物Rad51p和Dmc1p及其相互作用蛋白Rad54p和Tid1p的突变表型，发现它们在内含肽遗传中都起着关键作用。DSB末端加工蛋白Sae2p以及Mre11-Rad50-Xrs2复合物中的蛋白缺失也会导致归巢效率部分降低。与减数分裂重组一样，在内含肽归巢过程中经常观察到交叉事件。我们还观察到，羟基脲（HU）或clb5delta clb6delta突变导致的减数分裂前DNA复制缺失会减少VDE介导的DSB。
内含肽归巢中起作用的修复系统与Spo11p诱导的减数分裂重组共享分子机制。此外，与Spo11p诱导的DNA切割一样，减数分裂前DNA复制是VDE诱导DSB的先决条件。因此，VMA1内含肽利用参与减数分裂和重组过程的几种宿主因子来传播其遗传信息，并通过与生物体建立寄生关系来保证其后代。
