Funayama T, Narumi I, Kikuchi M, Kitayama S, Watanabe H, Yamamoto K
Biotechnology Laboratory, Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Japan.
Mutat Res. 1999 Oct 22;435(2):151-61. doi: 10.1016/s0921-8777(99)00044-0.
We isolated a radiosensitive mutant strain, KR4128, from a wild-type strain of Deinococcus radiodurans, which is known as a extremely radioresistant bacterium. The gene that restore the defect of the mutant in DNA repair was cloned, and it turned out to be the homolog of the recN gene of Escherichia coli. The recN gene encoded a protein of 58 kDa, and, in its N-terminal region, a potential ATP binding domain was conserved as expected for a prokaryotic RecN protein. An analysis of sequence of the mutant recN gene revealed a G:C to T:A transversion near the 3' end of the coding region. This alteration causes an ochre mutation, and results in the truncation of 47 amino acids from the C-terminal region of the RecN protein. The null mutant of recN gene was constructed by insertional mutagenesis, and it showed substantial sensitivities to various types of DNA damaging agents, indicating that a single defect in the recN gene can directly affect the DNA damage resistant phenotype in D. radiodurans. The recN locus of KR4128 was also disrupted and the disruptant indicated the sensitivity that was indistinguishable from its progenitor. The result indicate that the transversion in the recN gene of KR4128 cells causes a complete loss of function of the RecN protein and thus the C-terminal region of the RecN protein includes domain essential to its function.
我们从耐辐射球菌的野生型菌株中分离出了一个辐射敏感突变株KR4128，耐辐射球菌是一种已知的极具辐射抗性的细菌。克隆了能够修复该突变体DNA修复缺陷的基因，结果发现它是大肠杆菌recN基因的同源物。recN基因编码一个58 kDa的蛋白质，并且在其N端区域，一个潜在的ATP结合结构域如预期的原核RecN蛋白一样保守。对突变的recN基因序列分析显示，在编码区3'端附近发生了G:C到T:A的颠换。这种改变导致了赭石型突变，并导致RecN蛋白C端区域截短47个氨基酸。通过插入诱变构建了recN基因的缺失突变体，它对各种类型的DNA损伤剂表现出显著的敏感性，表明recN基因的单一缺陷可直接影响耐辐射球菌的抗DNA损伤表型。KR4128的recN位点也被破坏，破坏体表现出与其亲本无法区分的敏感性。结果表明，KR4128细胞recN基因中的颠换导致RecN蛋白功能完全丧失，因此RecN蛋白的C端区域包含其功能所必需的结构域。
