DNA repair of clustered uracils in HeLa cells.
Two or more base damages, abasic sites or single-strand breaks (SSBs) within two helical turns of the DNA form a multiply damaged site (MDS) or clustered lesion. Studies in vitro and in bacteria indicate that attempts to repair two closely opposed base lesions can potentially form a lethal double-strand break (DSB). Ionizing radiation and chemotherapeutic agents introduce complex lesions, and the inability of a cell to repair MDSs is believed to contribute to the lethality of these treatments. The goal of this work was to extend the in vitro studies by examining MDS repair in mammalian cells under physiological conditions. Here, two opposing uracil residues separated by 3, 5, 7, 13 or 29 base-pairs were chosen as model DNA lesions. Double-stranded oligonucleotides containing no damage, a single uracil residue or the MDS were introduced into a non-replicating mammalian construct within the firefly luciferase open reading frame, or at the 5' or 3' end of the luciferase expression cassette. Following transient transfection into HeLa cells, luciferase activity was measured or plasmid DNA was re-isolated from the cells. Formation of a DSB was expected to decrease luciferase expression. However, certain single uracil residues as well as the MDSs decreased luciferase activity, which suggested that the reduction in activity was not due to DSB formation. In fact, Southern analysis of the re-isolated plasmid did not show the presence of linear DNA and demonstrated that none of the constructs was destroyed during repair. Further analysis of the re-isolated DNA demonstrated that only a small percentage of molecules originally carrying a single lesion or an MDS contained deletions. This work indicates that the majority of the clustered lesions were not converted to DSBs and that repair systems in mammalian cells may have established mechanisms to avoid the accumulation of SSB-repair intermediates.
Malyarchuk
Svitlana
S
Department of Molecular and Cellular Physiology, Louisiana Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130, USA.
Harrison
Lynn
L
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004260:DNA Repair; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014498:Uracil / Q000378:metabolism*
D014498:Uracil; D004247:DNA; D049409:Luciferases, Firefly