Roles of uracil-DNA glycosylase and apyrimidinic endonucleases in the molecular 5-bromo-2'-deoxyuridine photosensitization in Escherichia coli K-12.
The molecular mechanism for 5-bromo-2'-deoxyuridine (BrdU) photosensitization was studied in thymine-requiring wild-type and uracil-DNA glycosylase (UDG)-deficient ung mutant cells of Escherichia coli K-12. Wild-type cells were more sensitive to BrdU photosensitization than ung mutant cells. UV induced the identical numbers of alkaline sucrose single-strand breaks (SSB) in 5-bromouracil-DNA (BrU-DNA) of both the wild type and ung mutant. The ung mutant cells repaired SSB almost completely, whereas the wild-type cells with UDG produced more adverse SSB by 90 min after UV. Neutral agarose gel electrophoresis of minipreps indicated that UV induced (1) more smears of host BrU-DNA possibly by more double-strand breaks (DSB) and (2) a greater decline of pBR322 Form I BrU-DNA in the wild-type cells than the ung cells. These results indicated a greater induction of SSB by apyrimidinic (AP) endonucleases in wild-type cells. The ung/wild ratios (= 1.7-1.9) for cellular and plasmid BrdU sensitizations after growth in 50% BrdU were similar. The extents of UDG-dependent and UDG-independent sensitizations in wild-type cells were approximately 40 and approximately 60%, respectively. The xth nfo double mutant defective in both exonuclease III and endonuclease IV was more sensitive to BrdU photosensitization than the wild type, indicating that an excess of AP sites remaining after uracil excision in the xth nfo mutant causes a greater BrdU photosensitization than SSB by AP endonucleases in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Yamamoto
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Department of Radiation Biophysics and Genetics, Kobe University School of Medicine, Japan.
Fujiwara
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Y
D001973:Bromodeoxyuridine / Q000494:pharmacology*; D045647:DNA Glycosylases; D004269:DNA, Bacterial / Q000187:drug effects / Q000528:radiation effects; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000528:radiation effects; D029968:Escherichia coli Proteins; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D017319:Photosensitizing Agents / Q000494:pharmacology; D014466:Ultraviolet Rays; D051981:Uracil-DNA Glycosidase
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D017319:Photosensitizing Agents; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D001973:Bromodeoxyuridine