Krishna T Hari, Mahipal S, Sudhakar A, Sugimoto H, Kalluri Raghu, Rao K Subba
ICMR-Advanced Center for Research on Aging and Brain, Department of Biochemistry, University of Hyderabad, Hyderabad, India.
J Neurochem. 2005 Feb;92(4):818-23. doi: 10.1111/j.1471-4159.2004.02923.x.
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.
含有1个和4个核苷酸短间隙的合成脱氧寡聚双链体被用作模型底物，以评估从不同年龄大鼠大脑皮层制备的神经元提取物的DNA间隙修复能力。我们的结果表明，神经元中的间隙修复活性随年龄显著降低。通过用纯重组大鼠肝DNA聚合酶β补充神经元提取物，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，该活性基本上通过以缓慢的分布链置换模式添加核苷酸来进行，以达到完整的模板长度（32聚体）。然而，在较低浓度的DNA聚合酶β下，间隙修复通过间隙填充然后连接到下游引物快速发生，这是一种节能方式。为此，所需的条件是下游引物上存在5'-PO4，并且除了重组DNA聚合酶β之外，还用DNA连接酶补充衰老的神经元提取物。这些结果表明，衰老神经元由于DNA聚合酶β和DNA连接酶的缺乏而无法影响碱基切除修复（BER），用这两种因子强化衰老的神经元提取物可以恢复丧失的BER活性。
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