Maryon E, Carroll D
Department of Cellular, Viral and Molecular Biology, University of Utah School of Medicine, Salt Lake City 84132.
Mol Cell Biol. 1989 Nov;9(11):4862-71. doi: 10.1128/mcb.9.11.4862-4871.1989.
Linear DNA injected into Xenopus laevis oocyte nuclei recombines with high efficiency if homologous sequences are present at overlapping molecular ends. We found that injected linear DNA was degraded by a 5'----3' strand-specific exonuclease activity during incubation in the oocyte nucleus to leave a heterogeneous population of 3'-tailed molecules. Decreasing the concentration of DNA injected increased the heterogeneity and the average rate of degradation. The 3' tails created were relatively stable; among molecules persisting after overnight incubation, many had 3' tails intact to within 10 bases of the original ends. DNA molecules that were efficient substrates for homologous recombination in oocytes were also partially degraded, leaving 3' tails. We found no evidence for other potent nuclease activities. If molecules with recessed 3'-OH ends were injected, endogenous polymerase efficiently resynthesized complementary strands before degradation of the 5' tails occurred. 3'-tailed molecules are plausible intermediates in the initiation of homologous recombination events in Xenopus oocyte nuclei.
如果在重叠的分子末端存在同源序列,注射到非洲爪蟾卵母细胞核中的线性DNA会高效重组。我们发现,注射的线性DNA在卵母细胞核中孵育期间会被一种5'→3'链特异性核酸外切酶活性降解,从而留下一群异质的3'尾分子。降低注射的DNA浓度会增加异质性和平均降解速率。产生的3'尾相对稳定;在过夜孵育后仍存在的分子中,许多分子的3'尾在原始末端的10个碱基范围内保持完整。在卵母细胞中作为同源重组有效底物的DNA分子也会被部分降解,留下3'尾。我们没有发现其他强效核酸酶活性的证据。如果注射具有凹陷3'-OH末端的分子,内源性聚合酶会在5'尾降解之前有效地重新合成互补链。3'尾分子可能是非洲爪蟾卵母细胞核中同源重组事件起始的中间体。