Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, MS 400 085, India.
Biochem Cell Biol. 2010 Oct;88(5):783-90. doi: 10.1139/o10-075.
A multiprotein DNA processing complex isolated from Deinococcus radiodurans contains the DNA repair protein PprA, an ATP-type DNA repair ligase (LigB) encoded by the drB0100 gene, and protein kinase activity. An ATP-dependent DNA end-joining activity was detected in the complex. To elucidate the function of the drB0100 gene, we generated the deletion mutant for the DR_B0100 ORF. The mutant exhibited a nearly 2-log cycle reduction in growth rate when exposed to a 10,000 Gray dose of γ-radiation, and a significant loss in mitomycin C and methylmethane sulphonate tolerance as compared with wild type. Functional complementation of these phenotypes required the wild-type copy of drB0100 along with other genes such as drb0099 and drb0098, organized downstream in the operon. The in vitro DNA ligase activity of LigB was stimulated severalfold by PprA in the presence of the recombinant DRB0098 protein. However, this activity did not improve when PprA was substituted with purified DRB0099 protein or when DRB0098 protein was substituted with the DRB0099 protein in the presence of PprA in solution. These results suggest that PprA and DRB0098 protein are required for LigB function. Furthermore, they also suggest that the LigB operon components contribute to radiation resistance and double-strand break (DSB) repair in D. radiodurans.
从耐辐射球菌中分离得到的一种多蛋白 DNA 加工复合物含有 DNA 修复蛋白 PprA、由 drB0100 基因编码的 ATP 型 DNA 修复连接酶（LigB）和蛋白激酶活性。在该复合物中检测到 ATP 依赖性 DNA 末端连接活性。为了阐明 drB0100 基因的功能，我们生成了该基因的缺失突变体。与野生型相比，突变体在暴露于 10000 Gray 剂量的γ射线时的生长速度降低了近 2 个对数周期，并且对丝裂霉素 C 和甲基甲烷磺酸的耐受性显著降低。这些表型的功能互补需要野生型 drB0100 及其它基因，如 drb0099 和 drb0098，这些基因在操纵子中排列在下游。在存在重组 DRB0098 蛋白的情况下，PprA 将 LigB 的体外 DNA 连接酶活性提高了几倍。然而，当用纯化的 DRB0099 蛋白替代 PprA 时，或者当在溶液中用 DRB0099 蛋白替代 PprA 时，这种活性并没有改善。这些结果表明 PprA 和 DRB0098 蛋白是 LigB 功能所必需的。此外，它们还表明 LigB 操纵子成分有助于耐辐射球菌的辐射抗性和双链断裂（DSB）修复。
