Molecular characterization of genes involved in DNA repair in Xanthomonas campestris pv. citri

• Main Authors: Yuan-Chang Yang, 楊源昌
• Other Authors: Jenn Tu
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/61735235490535887093

博士 === 國防醫學院 === 生命科學研究所 === 89 === Abstract The recA gene of Xanthomonas campestris pathovar citri (X. c. pv. citri) was cloned and sequenced. The nucleotide sequence analysis revealed an open reading frame of 1032 bp that encodes a 344-amino acid protein and that shows a high degree of homology to recA genes from other bacteria. Two additional open reading frames were identified in the recA region of the X. c. pv. citri genome: one located immediately upstream from recA that encodes a 213-amino acid protein with a high degree of similarity to the LexA protein of E. coli and another located immediately downstream of recA that encodes a 162-amino acid protein with sequence similarity to the RecX protein of P. aeruginosa. The transcription start site of lexA and recA, respectively were identified, and the upstream region of these genes were shown to confer sensitivity to DNA-damaging agents on a luciferase report gene construct. The level of recA expression in a lexA mutant is similar to that from wild type cells expressed to a DNA-damaging agent, indicating that LexA represses the expression of recA gene in X. c. pv. citri. Immunoblot analysis also revealed that the overexpressed LexA protein functions as a repressor of recA expression in X. c. pv. citri, and that the mitomycin C-induced increase in the abundance of RecA was accompanied by specific proteolysis of LexA that required RecA. Overexpression of X. c. pv. citri LexA resulted in an increased sensitivity of cells to DNA-damaging agents, mitomycin C and ultraviolet radiation, in X. c. pv. citri and E. coli, indicating that the recombinant X. c. pv. citri LexA protein was functional in a different bacterial species. Gel retardation assays demonstrated that X. c. pv. citri LexA interacts with the promoter region of X. c. pv. citri recA and recX as well as lexA gene. DNase I footprinting of the lexA promoter showed that the TTAGTAGTAATACTACTAA sequence is required for the function of the DNA-protein complex. Mutational analysis of that TTAG-N11-CTAA and TTAG-N8-CGAA sequences are necessary for LexA binding. These results indicate that X. c. pv. citri LexA functions as a repressor of gene expression by binding to the region different from that of E. coli.