Removal of deoxyinosine from the Escherichia coli chromosome as studied by oligonucleotide transformation.
Deoxyinosine (dI) is produced in DNA by the hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the product of the nfi gene. The repair was studied in vivo using high-efficiency oligonucleotide transformation mediated by the Beta protein of bacteriophage lambda in a mismatch repair-deficient host. Escherichia coli was transformed with oligonucleotides containing a selectable A-G base substitution mutation. When the mutagenic dG was replaced by a dI in the oligonucleotide, it lost 93-99% of its transforming ability in an nfi(+) cell, but it remained fully functional in an nfi mutant. Therefore, endonuclease V is responsible for most of the removal of deoxyinosine from DNA. New nfi mutants were isolated based on the strong selection provided by their tolerance for transformation by dI-containing DNA. The repair patch for dI was then measured by determining how close to the transforming dG residue a dI could be placed in the oligonucleotide before it interferes with transformation. At the endonuclease V cleavage site, three nucleotides were preferentially removed from the 3' end and two nucleotides were removed from the 5' end. dI:dT and dI:dC base pairs gave the same results. Caveats include possible interference by Beta protein and by mispaired bases. Thus, oligonucleotide transformation can be used to determine the relative importance of redundant repair pathways, to isolate new DNA repair mutants, and to determine with high precision the sizes of repair tracts in intact cells.
Weiss
Bernard
B
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Whitehead Building, Atlanta, GA 30322, USA. bweiss2@emory.edu
D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007288:Inosine / Q000031:analogs & derivatives* / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D009841:Oligonucleotides; D007288:Inosine; D043224:Deoxyribonuclease (Pyrimidine Dimer); C012271:deoxyinosine