Lehman C W, Carroll D
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10840-4. doi: 10.1073/pnas.88.23.10840.
Xenopus laevis oocytes efficiently recombine linear DNA injected into their nuclei (germinal vesicles). This process requires homologous sequences at or near the molecular ends. Here we report that a cell-free extract made from germinal vesicles is capable of accomplishing the complete recombination reaction in vitro. Like the in vivo process, the extract converts the overlapping ends of linear substrate molecules into covalently closed products. Establishment of this cell-free system has allowed examination of the cofactors required for recombination. The first step involves a 5'----3' exonuclease activity that requires a divalent cation but not NTPs. Completion of recombination requires a hydrolyzable NTP; maximal product formation occurs in the presence of millimolar levels of ATP or dATP. At submillimolar levels of all four dNTPs, homologous recombination is inefficient, and a side reaction produces end-joined products. This cell-free system should facilitate a step-by-step understanding of an homologous recombination pathway that operates not only in Xenopus laevis oocytes but also in cells from a wide variety of organisms.
非洲爪蟾卵母细胞能够高效地重组注入其细胞核（生发泡）中的线性DNA。这一过程在分子末端或其附近需要同源序列。在此我们报道，从生发泡制备的无细胞提取物能够在体外完成完整的重组反应。与体内过程一样，提取物将线性底物分子的重叠末端转化为共价闭合产物。这个无细胞系统的建立使得对重组所需辅助因子的研究成为可能。第一步涉及一种5'→3'核酸外切酶活性，该活性需要二价阳离子但不需要NTP。重组的完成需要一种可水解的NTP；在毫摩尔水平的ATP或dATP存在下会出现最大产物形成。在所有四种dNTP的亚毫摩尔水平下，同源重组效率低下，并且会发生一个副反应产生末端连接产物。这个无细胞系统应该有助于逐步理解不仅在非洲爪蟾卵母细胞中而且在来自多种生物体的细胞中起作用的同源重组途径。
