Fortier Sean, Yang Xiaojie, Wang Yi, Bennett Richard A O, Strauss Phyllis R
Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Biochemistry. 2009 Jun 16;48(23):5396-404. doi: 10.1021/bi900253d.
The base excision repair (BER) pathway recognizes and repairs most nonbulky lesions, uracil and abasic (AP) sites in DNA. Several participants are embryonic lethals in knockout mice. Since the pathway has never been investigated during embryogenesis, we characterized the first three steps of BER in zebrafish extracts from unfertilized eggs, embryos at different developmental stages, and adults. Using a 45-mer double-stranded substrate with a U/G mispair at position 21, we showed that extracts from all stages are capable of performing BER. Before 3 days postfertilization (dpf), aphidicolin-sensitive polymerases perform most nucleotide insertion. In fact, eggs and early stage embryos lack DNA polymerase-beta protein. After the eggs have hatched at 3 dpf, an aphidicolin-resistant polymerase, probably DNA polymerase-beta, becomes the primary polymerase. Previously, we showed that when the zebrafish AP endonuclease protein (ZAP1) level is knocked down, embryos cease dividing after the initial phase of rapid proliferation and die without apoptosis shortly thereafter. Nevertheless, extracts from embryos in which ZAP1 has been largely depleted process substrate as well as extracts from control embryos. Since apex1 and apex2 are both strongly expressed in early embryos relative to adults, these data indicate that both may play important roles in DNA repair in early development. In brief, the major differences in BER performed by early stage embryos and adults are the absence of DNA polymerase-beta, leading to predominance of replicative polymerases, and the presence of backup Mg(2+)-dependent endonuclease activity in early stage embryos. The switch to normal, adult BER occurs fully when the embryos hatch from the chorionic membrane and encounter normal oxidative stress.
碱基切除修复（BER）途径可识别并修复大多数非庞大损伤、DNA中的尿嘧啶和无碱基（AP）位点。该途径的几个参与因子在基因敲除小鼠中是胚胎致死性的。由于该途径在胚胎发生过程中从未被研究过，我们对来自未受精卵、不同发育阶段的胚胎以及成体斑马鱼提取物中的BER的前三个步骤进行了表征。使用在第21位有U/G错配的45聚体双链底物，我们表明所有阶段的提取物都能够进行BER。在受精后3天（dpf）之前，对阿非科林敏感的聚合酶进行大多数核苷酸插入。事实上，卵和早期胚胎缺乏DNA聚合酶β蛋白。在3 dpf卵孵化后，一种对阿非科林有抗性的聚合酶，可能是DNA聚合酶β，成为主要聚合酶。此前，我们表明当斑马鱼AP核酸内切酶蛋白（ZAP1）水平被敲低时，胚胎在快速增殖的初始阶段后停止分裂，并在此后不久无凋亡地死亡。然而，ZAP1已被大量消耗的胚胎提取物处理底物的能力与对照胚胎提取物相同。由于相对于成体，apex1和apex2在早期胚胎中均强烈表达，这些数据表明两者可能在早期发育中的DNA修复中发挥重要作用。简而言之，早期胚胎和成体进行BER的主要差异在于早期胚胎中缺乏DNA聚合酶β，导致复制性聚合酶占主导，以及早期胚胎中存在备用的Mg(2+)依赖性核酸内切酶活性。当胚胎从绒毛膜孵化并遇到正常氧化应激时，完全转变为正常的成体BER。
