PDF(Pubmed)
Abstract：
Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3\'-to-5\' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in ∆nei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of ∆lhr cells by mutant lhr alleles depended on Lhr\'s ATPase/helicase activities and its tetrameric quaternary structure. The lhr-nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.IMPORTANCEThe DNA inter-strand crosslinking agents mitomycin C, cisplatin, and psoralen-UVA are used clinically for the treatment of cancers and skin diseases; they have been invaluable in elucidating the pathways of inter-strand crosslink repair in eukaryal systems. Whereas DNA crosslinkers are known to trigger a DNA damage response in bacteria, the roster of bacterial crosslink repair factors is incomplete and likely to vary among taxa. This study implicates the DNA damage-inducible mycobacterial lhr-nei2 gene operon in protecting Mycobacterium smegmatis from killing by inter-strand crosslinkers. Whereas interdicting the activity of the Lhr helicase sensitizes mycobacteria to mitomycin C, cisplatin, and psoralen-UVA, the Nei2 glycosylase functions uniquely in evasion of damage caused by psoralen-UVA.
摘要：
耻垢分枝杆菌Nei2是一种单体酶，对单链DNA具有APβ-裂解酶活性。Nei2及其操纵子邻居Lhr（四聚体3'至5'解旋酶）的表达，在暴露于DNA损伤剂的分枝杆菌中诱导。这里,我们发现，nei2缺失使耻垢分枝杆菌对DNA链间交联剂三甲基补骨脂素的杀伤敏感，但对交联剂丝裂霉素C和顺铂的杀伤不敏感。相比之下,lhr的缺失对所有三种交联剂的杀伤敏感。我们报告了重组Nei2的1.45µ晶体结构，该结构由位于适合DNA结合的中央凹槽两侧的N和C末端叶组成。C叶包括四半胱氨酸锌络合物。突变分析鉴定了N末端脯氨酸残基（ORF的Pro2）和Lys51，但不是Glu3，是AP裂解酶活性所必需的。我们发现Nei2在单链DNA上具有5-羟基尿嘧啶糖基化酶活性，该活性被Glu3和Lys51的丙氨酸突变而不是Pro2的丙氨酸突变所消除。通过κnei2细胞中野生型和突变型nei2等位基因的表达来测试补骨脂素敏感性，确定AP裂解酶活性对于交联修复既不足够也不重要。相比之下,突变型lhr等位基因对Δlhr细胞补骨脂素敏感性的补充取决于Lhr的ATP酶/解旋酶活性及其四聚体四级结构。lhr-nei2操纵子包含独特的细菌系统以纠正链间交联。重要DNA链间交联剂丝裂霉素C，顺铂,补骨脂素-UVA在临床上用于治疗癌症和皮肤病;它们在阐明真核系统中链间交联修复的途径方面具有非常重要的价值。尽管已知DNA交联剂会引发细菌中的DNA损伤反应，细菌交联修复因子的花名册是不完整的，并且可能在分类单元之间有所不同。这项研究暗示DNA损伤可诱导的分枝杆菌lhr-nei2基因操纵子可以保护耻垢分枝杆菌免受链间交联剂的杀死。尽管阻断Lhr解旋酶的活性会使分枝杆菌对丝裂霉素C敏感，顺铂,和补骨脂素-UVA,Nei2糖基化酶在避免补骨脂素-UVA引起的损伤中起独特的作用。
