Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
DNA Repair (Amst). 2010 Jan 2;9(1):66-75. doi: 10.1016/j.dnarep.2009.10.009. Epub 2009 Nov 26.
The mechanism by which double-strand DNA breaks are repaired in the radiation-resistant bacterium Deinococcus radiodurans is not well understood. This organism lacks the RecBCD helicase/nuclease, which processes broken DNA ends in other bacteria. The RecF pathway is an alternative pathway for recombination and DNA repair in E. coli, when RecBCD is absent due to mutation, and D. radiodurans may rely on enzymes of this pathway for double-strand break repair. The RecJ exonuclease is thought to process broken DNA ends for the RecF pathway. We attempted to delete the recJ gene from D. radiodurans, using homologous recombination to replace the gene with a streptomycin-resistance cassette. We were unable to obtain a complete deletion mutant, in which the gene is deleted from all of the chromosome copies in this polyploid organism. Quantitative real-time PCR shows that the heterozygous mutants have a recJ gene copy that is ca. 10-30% that of the wild-type. Mutants with reduced recJ gene copy grow slowly and are more sensitive than wild-type to UV irradiation, gamma irradiation, and hydrogen peroxide. The mutants are as resistant as wild-type to methyl-methanesulfonate. The D. radiodurans RecJ protein was expressed in E. coli and purified under denaturing conditions. The re-folded protein has nuclease activity on single-stranded DNA with specificity similar to that of E. coli RecJ exonuclease.
耐辐射球菌中双链 DNA 断裂修复的机制尚未完全阐明。该生物体缺乏 RecBCD 解旋酶/核酸酶，该酶在其他细菌中处理断裂的 DNA 末端。在大肠杆菌中，当因突变而缺乏 RecBCD 时，RecF 途径是一种替代的重组和 DNA 修复途径，而耐辐射球菌可能依赖该途径的酶来修复双链断裂。RecJ 核酸外切酶被认为可用于 RecF 途径处理断裂的 DNA 末端。我们试图使用同源重组将链霉素抗性盒取代耐辐射球菌中的 recJ 基因，从而从耐辐射球菌中删除 recJ 基因。我们无法获得完全缺失突变体，在这种多倍体生物中，该基因从所有染色体拷贝中缺失。实时定量 PCR 显示，杂合突变体的 recJ 基因拷贝数约为野生型的 10-30%。基因拷贝数减少的突变体生长缓慢，比野生型对紫外线、γ射线和过氧化氢更敏感。突变体对甲基甲磺酸的抗性与野生型相同。耐辐射球菌 RecJ 蛋白在大肠杆菌中表达，并在变性条件下纯化。复性后的蛋白对单链 DNA 具有核酸酶活性，特异性与大肠杆菌 RecJ 核酸外切酶相似。
