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Effects of antitumour chemotherapics on the expression of genes of the nucleotide excision repair system in Escherichia coli
Irineu, Roberto - Silva1,2; von Kruger, Wanda2; Felício, Deise1; Vidal, Leonardo1; Cardoso, Angélica3; Britto, Constança3; Lage, Claudia1
1 Laboratório de Radiobiologia Molecular (IBCCF)
2 Unidade Multidisciplinar Genômica (IBCCF)
3 Laboratório de Biologia Molecular e Doenças Endêmicas (DBBM – Fiocruz)
Nucleotide Excision Repair (NER) is one of the processes of “revision” present virtually in all organisms to ensure DNA integrity. Considering the importance of this pathway on the process of removing the nucleotides bound to the antitumoural chemotherapics, we decided to observe the expression of NER genes in Escherichia coli after treatment with some of those drugs, namely, cisplatin, mitomicin-C and nitrogenous mustard. In the first segment of this work, the promotor regions of uvrA and uvrB genes were cloned in an expression vector upstream lacZ, possibiliting to estimate their transcriptional activities, by the level β-galactosidase produced.
Our results showed activation of the expression of both, uvrA and uvrB promoters particularly by the mitomicin treatment. The P3 uvrB promoter seems to have a crucial role in this process. Additionally, an interesting result is that P3 uvrB promoter apparently works as a repressor on basal transcription. Finally, proficient strains in both, NER pathway repair and SOS response, were submitted to quantitative analysis of transcripts associated to uvrABC, cho (encodes a UvrC-like) and recA genes by quantitative Real Time PCR (SyBr Green Methodology). Our results showed increased transcription of uvrA, B and C, cho and recA in the treated cells. However, when the above results are compared to those of survival of the NER mutants upon treatment with the same drugs, we concluded that the level of involvement of the NER proteins in the repair of the DNA depends on the drug used to generate the lesion. Interestingly, UvrB, a protein structurally and functionally homologous to the human XPD, was the only one indispensable for the removal and subsequent correction of lesions generated by all the three antitumour chemotherapics studied.
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