Overexpression of the natural recO sequence and its effects on DNA repair of Escherichia coli.
The recO gene is required for the RecF pathway of recombination and the repair of DNA daughter-strand gaps in Escherichia coli. In this work, the structural portion of recO was synthesized by the polymerase chain reaction (PCR) and cloned onto expression vectors at their Nco1 fusion cloning site, to eliminate the presence of mRNA leader sequence. While the plasmid carrying a Ptac promoter failed to overproduce RecO, the plasmid carrying a T7O10 promoter overproduced RecO in large quantity, indicating that the natural recO may be overexpressed. An increase of intracellular RecO, which may be due to the increased recO gene copies or to the induction of RecO synthesis, increased the UV resistance of recA, recF, and ssb cells, but did not increase the UV resistance of uvrB, uvrB recF, uvrB recA and uvrB ssb cells. We suggest that an increase of intracellular RecO may allow some recombination-deficient cells to perform more excision repair, thus increasing the survival. The possible causes for RecO overproduction on excision repair, and for the differential expression of recO by the Ptac and T7 promoter plasmids are discussed.
Department of Molecular and Cellular Biology, Chang Gung College of Medicine and Technology, Kwei-San, Tao-Yuan, Taiwan.
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CL
Yang
Y H
YH
Liu
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TC
Wang
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D017123:Bacteriophage T7; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004260:DNA Repair / Q000235:genetics*; D004269:DNA, Bacterial / Q000528:radiation effects; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011836:Radiation Tolerance / Q000235:genetics*; D011995:Recombination, Genetic; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D015345:Oligonucleotide Probes; C081006:RecO protein, E coli