Reduced DNA gap repair in aging rat neuronal extracts and its restoration by DNA polymerase β and DNA-ligase
Reduced DNA gap repair in aging rat neuronal extracts and its restoration by DNA polymerase β and DNA-ligase
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DOI：
10.1111/j.1471-4159.2004.02923.x
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发表时间：
2005-02-01
影响因子：
4.7
通讯作者：
Rao, KS
中科院分区：
文献类型：
--
作者：
Krishna, TH;Mahipal, S;Rao, KS
合成的脱氧寡核苷酸双链体含有1和4个核苷酸的短缺口作为模型底物，以评估从不同年龄的大鼠大脑皮层制备的神经元提取物的DNA缺口修复能力。我们的研究结果表明，神经元中的间隙修复活性随着年龄的增长而显着下降。通过补充纯重组大鼠肝DNA聚合酶β的神经元提取物，可以恢复降低的活性。高水平的DNA聚合酶β补充导致间隙填充活性，其基本上通过缓慢分布链置换模式添加核苷酸来进行，以实现完整的模板长度（32聚体）。然而，在较低浓度的DNA聚合酶β下，差距修复以能量有效的方式通过缺口填充随后连接到下游引物而快速发生。为了实现这一点，所需的条件是在下游引物上存在5 '-PO 4，以及除了重组DNA聚合酶β之外，还用DNA连接酶补充老化神经元提取物。这些结果表明，由于DNA聚合酶β和DNA连接酶的缺乏，衰老神经元无法影响碱基切除修复（BER），用这两种因子强化衰老神经元提取物可以恢复失去的BER活性。
Synthetic deoxy-oligo duplexes containing short gaps of 1 and 4 nucleotides were used as model substrates to assess the DNA gap repair ability of the neuronal extracts prepared from cerebral cortex of rats of different ages. Our results demonstrate that gap repair activity in neurons decreases markedly with age. The decreased activity could be restored by supplementing the neuronal extracts with pure recombinant rat liver DNA polymerase beta. High levels of DNA polymerase beta supplementation resulted in gap-filling activity that proceeded essentially through addition of nucleotides through a slow distributive strand displacement mode to achieve full template length (32-mer). However, at lower concentrations of DNA polymerase beta, the gap repair takes place quickly through gap filling followed by ligation to downstream primer, in an energy efficient manner. For this to happen, the conditions required are the presence of 5'-PO4 on the downstream primer and supplementation of aging neuronal extracts with DNA-ligase in addition to recombinant DNA polymerase beta. These results demonstrate that aging neurons are unable to affect base excision repair (BER) due to deficiency of DNA polymerase beta and DNA-ligase and fortifying aged neuronal extracts with these two factors can restore the lost BER activity.