Closely opposed apurinic/apyrimidinic sites are converted to double strand breaks in Escherichia coli even in the absence of exonuclease III, endonuclease IV, nucleotide excision repair and AP lyase cleavage.
Multiply damaged sites (MDSs) consist of two or more damages within 20 base pairs (bps) and are introduced into DNA by ionizing radiation. Using a plasmid assay, we previously demonstrated that repair in Escherichia coli generated a double strand break (DSB) from two closely opposed uracils when uracil DNA glycosylase initiated repair. To identify the enzymes that converted the resulting apurinic/apyrimidinic (AP) sites to DSBs, repair was examined in bacteria deficient in AP site cleavage. Since exonuclease III (xth) and endonuclease IV (nfo) mutant bacteria were able to introduce DSBs at the MDSs, we generated unique bacterial mutants deficient in UvrA, Xth and Nfo. However, the additional disruption of nucleotide excision repair (NER) did not prevent DSB formation. xth- nfo- nfi- bacteria also converted the MDSs to DSBs, ruling out endonuclease V as the candidate AP endonuclease. By using MDSs containing tetrahydrofuran (an AP site analog), it was determined that even in the absence of Xth, Nfo, NER and AP lyase cleavage, DSBs were formed from closely opposed AP sites. This finding implies that there is an unknown enzyme/repair pathway for MDSs, and multiple underlying repair systems in cells that can process closely opposed DNA damage into lethal lesions following exposure to ionizing radiation.
Harrison
Lynn
L
Department of Molecular and Cellular Physiology, Louisiana Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA. lclary@lsuhsc.edu
Brame
Katherine L
KL
Geltz
Laura E
LE
Landry
April M
AM
D001665:Binding Sites; D004247:DNA / Q000235:genetics*; D004249:DNA Damage / Q000528:radiation effects*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000172:deficiency* / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced) / Q000172:deficiency*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D029968:Escherichia coli Proteins; D011687:Purines / Q000737:chemistry*; D011740:Pyrimidine Dimers; D011743:Pyrimidines / Q000737:chemistry*; D014466:Ultraviolet Rays
D029968:Escherichia coli Proteins; D011687:Purines; D011740:Pyrimidine Dimers; D011743:Pyrimidines; D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase