Department of Molecular Biology, School of Biological Sciences, Centre for Excellence in Genomic Sciences, Madurai Kamaraj University (University with Potential for Excellence), Madurai 625021, Tamil Nadu, India.
DNA Repair (Amst). 2012 Nov 1;11(11):915-25. doi: 10.1016/j.dnarep.2012.09.005. Epub 2012 Oct 9.
An unconventional DNA repair termed SIR (SOS Independent Repair), specific to mitomycin C (MMC) damage elicited by a combination of specific Rif(R) (rpoB87) and Nal(R) (gyrA87) mutations in SOS un-inducible strains of Escherichia coli was reported by Kumaresan and Jayaraman (1988). We report here that the rpoB87 mutation defines a C(1565)→T(1565) transition changing S(522)→F(522) and gyrA87 defines a G(244)→A(244) transition changing D(82)→N(82). The reconstructed lexA3 rpoB87 gyrA87 strain (DM49RN) exhibited resistance to MMC but not to UV as expected. When mutations in several genes implicated in SOS/NER were introduced into DM49RN strain, uvrB mutation alone decreased the MMC resistance and suppressed SIR phenotype. This was alleviated about two fold by a plasmid clone bearing the uvrB(+) allele. Neither SulA activity as measured based on filamentation and sulA::gfp fluorescence analyses nor the transcript levels of sulA as seen based on RT-PCR analyses indicate a change in sulA expression in DM49RN strain. However, uvrB transcript levels are increased with or without MMC treatment in the same strain. While the presence of lexA3 allele in a plasmid clone was found to markedly decrease the MMC resistance of the DM49RN strain, the additional presence of uvrB(+) allele in the same clone alleviated the suppression of MMC resistance by lexA3 allele to a considerable extent. These results indicate the increased expression of uvrB in the DM49RN strain is probably from the LexA dependent promoter of uvrB. The sequence analyses of various uvrB mutants including those isolated in this study using localized mutagenesis indicate the involvement of the nucleotide phosphate binding domain (ATPase domain) and the ATP binding domain and/or the DNA binding domain of the UvrB protein in the MMC repair in DM49RN. The possible involvement of UvrB protein in the MMC damage repair in DM49RN strain in relation to DNA repair is discussed.
一种称为 SIR（SOS 独立修复）的非常规 DNA 修复，特定于米托蒽醌（MMC）损伤，由 Rif（R）（rpoB87）和 Nal（R）（gyrA87）突变在 SOS 非诱导大肠杆菌菌株中组合引起，由 Kumaresan 和 Jayaraman（1988 年）报道。我们在这里报告 rpoB87 突变定义了 C（1565）→T（1565）转换，将 S（522）→F（522）和 gyrA87 定义为 G（244）→A（244）转换，将 D（82）→N（82）。重建的 lexA3 rpoB87 gyrA87 菌株（DM49RN）表现出对 MMC 的抗性，但如预期的那样对 UV 没有抗性。当将几个涉及 SOS/NER 的基因中的突变引入 DM49RN 菌株时，仅 uvrB 突变降低了 MMC 抗性并抑制了 SIR 表型。带有 uvrB(+)等位基因的质粒克隆缓解了约两倍。基于丝状体形成和 sulA::gfp 荧光分析测量的 SulA 活性，以及基于 RT-PCR 分析观察到的 sulA 转录水平均表明 DM49RN 菌株中 sulA 表达没有变化。然而，在相同菌株中，无论是否用 MMC 处理，uvrB 转录水平均增加。虽然在质粒克隆中存在 lexA3 等位基因发现明显降低了 DM49RN 菌株的 MMC 抗性，但在同一克隆中额外存在 uvrB(+)等位基因在很大程度上缓解了 lexA3 等位基因对 MMC 抗性的抑制。这些结果表明，DM49RN 菌株中 uvrB 的表达增加可能来自 uvrB 的 LexA 依赖性启动子。使用局部诱变对各种 uvrB 突变体（包括本研究中分离的突变体）进行的序列分析表明，UvrB 蛋白的核苷酸磷酸结合结构域（ATP 酶结构域）和 ATP 结合结构域和/或 DNA 结合结构域参与了 DM49RN 中的 MMC 修复。讨论了 UvrB 蛋白在 DM49RN 菌株中与 DNA 修复相关的 MMC 损伤修复中的可能作用。
