Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China.
DNA Repair (Amst). 2012 Apr 1;11(4):349-56. doi: 10.1016/j.dnarep.2011.11.008. Epub 2012 Jan 31.
The single-stranded DNA-specific nuclease RecJ is found in most bacteria where it is involved in the RecFOR double-stranded break (DSBs) repair pathway. DSBs repair mainly occurs via the RecFOR pathway in Deinococcus radiodurans, a well-known radiation-resistant bacterium. A recJ null mutant was constructed to investigate the role of recJ in D. radiodurans. recJ inactivation caused growth defects and sensitivity to high temperatures. However, the radiation resistance of the recJ mutant was only moderately decreased. The full-length D. radiodurans RecJ (DrRecJ) protein was expressed and purified to further characterize its biochemical properties. DrRecJ possessed a Mn(2+) concentration-dependent nuclease activity where the optimal Mn(2+) concentration was 0.1mM. DrRecJ had a similar activity profile after adding 10mM Mg(2+) to reactions with different Mn(2+) concentrations, indicating that Mn(2+) is a RecJ regulator. Escherichia coli RecJ has no activity on 5' ssDNA tails shorter than 6-nt, but DrRecJ could effectively degrade DNA with a 4-nt 5' ssDNA tail, suggesting that DrRecJ may have a wider range of DNA substrates. Moreover, SSB in D. radiodurans stimulated the DrRecJ exonuclease activity, whereas DdrB inhibited it and provided protection to ssDNA. Overall, our results indicate that recJ is a nonessential gene in D. radiodurans and that the activity of DrRecJ is regulated by Mn(2+) and SSB-DdrB.
单链 DNA 特异性核酸内切酶 RecJ 存在于大多数细菌中，在其中参与 RecFOR 双链断裂 (DSBs) 修复途径。DSBs 修复主要发生在抗辐射细菌 Deinococcus radiodurans 中的 RecFOR 途径中。构建了 recJ 缺失突变体以研究 recJ 在 D. radiodurans 中的作用。recJ 失活导致生长缺陷和对高温敏感。然而，recJ 突变体的辐射抗性仅适度降低。表达并纯化全长 D. radiodurans RecJ (DrRecJ) 蛋白以进一步表征其生化特性。DrRecJ 具有依赖 Mn(2+)浓度的核酸酶活性，最佳 Mn(2+)浓度为 0.1mM。在向不同 Mn(2+)浓度的反应中添加 10mM Mg(2+)后，DrRecJ 具有相似的活性谱，表明 Mn(2+)是 RecJ 的调节剂。大肠杆菌 RecJ 对短于 6-nt 的 5' ssDNA 尾巴没有活性，但 DrRecJ 可以有效降解具有 4-nt 5' ssDNA 尾巴的 DNA，表明 DrRecJ 可能具有更广泛的 DNA 底物。此外，D. radiodurans 中的 SSB 刺激 DrRecJ 外切核酸酶活性，而 DdrB 抑制它并为 ssDNA 提供保护。总体而言，我们的结果表明 recJ 是 D. radiodurans 中的非必需基因，并且 DrRecJ 的活性受 Mn(2+)和 SSB-DdrB 调节。
