Cheng Kaiying, Chen Xuanyi, Xu Guangzhi, Wang Liangyan, Xu Hong, Yang Su, Zhao Ye, Hua Yuejin
Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China.
Agriculture and Food Science School, Zhejiang Agriculture and Forestry University, Zhejiang, Lin'an, China.
J Bacteriol. 2015 Jun 15;197(12):2048-61. doi: 10.1128/JB.00018-15. Epub 2015 Apr 13.
In archaea, the NurA nuclease and HerA ATPase/helicase, together with the Mre11-Rad50 complex, function in 3' single-stranded DNA (ssDNA) end processing during homologous recombination (HR). However, bacterial homologs of NurA and HerA have not been characterized. From Deinococcus radiodurans, we identified the manganese-dependent 5'-to-3' ssDNA/double-stranded DNA (dsDNA) exonuclease/endonuclease NurA (DrNurA) and the ATPase HerA (DrHerA). These two proteins stimulated each other's activity through direct protein-protein interactions. The N-terminal HAS domain of DrHerA was the key domain for this interaction. Several critical residues of DrNurA and DrHerA were verified by site-directed mutational analysis. Temperature-dependent activity assays confirmed that the two proteins had mesophilic features, with optimum activity temperatures 10 °C to 15 °C higher than their optimum growth temperatures. Knocking out either nurA or herA affected cell proliferation by shortening the growth phase, especially for growth at a high temperature (37 °C). In addition, both mutant strains displayed almost 10-fold-reduced intermolecular recombination efficiency, indicating that DrNurA and DrHerA might be involved in homologous recombination in vivo. However, single- and double-gene deletions did not show significantly decreased radioresistance. Our results confirmed that the biochemical activities of bacterial NurA and HerA proteins were conserved with archaea. Our phenotypical results suggested that these proteins might have different functions in bacteria.
Deinococcus radiodurans NurA (DrNurA) was identified as a manganese-dependent 5'-to-3' ssDNA/dsDNA exonuclease/endonuclease, and Deinococcus radiodurans HerA (DrHerA) was identified as an ATPase. Physical interactions between DrNurA and DrHerA explained mutual stimulation of their activities. The N-terminal HAS domain on DrHerA was identified as the interaction domain. Several essential functional sites on DrNurA and DrHerA were characterized. Both DrHerA and DrNurA showed mesophilic biochemical features, with their optimum activity temperatures 10 °C to 15 °C higher than their optimum growth temperatures in vitro. Knockout of nurA or herA led to abnormal cell proliferation and reduced intermolecular recombination efficiency but no obvious effect on radioresistence.
在古菌中，NurA核酸酶和HerA ATP酶/解旋酶与Mre11-Rad50复合物一起，在同源重组（HR）过程中参与3'单链DNA（ssDNA）末端加工。然而，NurA和HerA的细菌同源物尚未得到表征。我们从耐辐射球菌中鉴定出了锰依赖性的5'至3' ssDNA/双链DNA（dsDNA）核酸外切酶/核酸内切酶NurA（DrNurA）和ATP酶HerA（DrHerA）。这两种蛋白质通过直接的蛋白质-蛋白质相互作用刺激彼此的活性。DrHerA的N端HAS结构域是这种相互作用的关键结构域。通过定点突变分析验证了DrNurA和DrHerA的几个关键残基。温度依赖性活性测定证实这两种蛋白质具有嗜温特性，其最佳活性温度比最佳生长温度高10℃至15℃。敲除nurA或herA会通过缩短生长阶段影响细胞增殖，尤其是在高温（37℃）下生长时。此外，两种突变菌株的分子间重组效率均降低了近10倍，这表明DrNurA和DrHerA可能参与体内同源重组。然而，单基因和双基因缺失并未显示出明显降低的抗辐射性。我们的结果证实细菌NurA和HerA蛋白与古菌的生化活性是保守的。我们的表型结果表明这些蛋白在细菌中可能具有不同的功能。
耐辐射球菌NurA（DrNurA）被鉴定为锰依赖性的5'至3' ssDNA/dsDNA核酸外切酶/核酸内切酶，耐辐射球菌HerA（DrHerA）被鉴定为ATP酶。DrNurA和DrHerA之间的物理相互作用解释了它们活性的相互刺激。DrHerA上的N端HAS结构域被鉴定为相互作用结构域。表征了DrNurA和DrHerA上的几个必需功能位点。DrHerA和DrNurA在体外均表现出嗜温生化特性，其最佳活性温度比最佳生长温度高10℃至15℃。敲除nurA或herA导致细胞增殖异常和分子间重组效率降低，但对抗辐射性无明显影响。
