Repair of uracil residues closely spaced on the opposite strands of plasmid DNA results in double-strand break and deletion formation

Abstract

The role of closely spaced lesions on both DNA strands in the induction of double-strand breaks and formation of deletions was studied. For this purpose a polylinker sequence flanked by 165 bp direct repeats was inserted within the tet gene of pBR327. This plasmid was used to construct DNA containing one or two uracil residues which replaced cytosine residues in the KpnI restriction site of the polylinker. Incubation of the plasmid DNA construct with Escherichia coli cell-free extracts showed that double-strand breaks occurred as a result of excision repair of the opposing uracil residues by uracil-DNA glycosylase (in extracts from ung+ but not in extracts from ung- E. coli strains). Recombination of direct repeats, induced by double-strand breakage of plasmid DNA, can lead to the deletion of the polylinker and of one of the direct repeats, thus restoring the tet+ gene function which can be detected by the appearance of tetracycline-resistant colonies of transformants. Transformation of E. coli cells with single or double uracil-containing DNAs demonstrated that DNA containing two closely spaced uracil residues was tenfold more effective in the induction of deletions than DNA containing only a single uracil residue. The frequency of deletions is increased tenfold in an ung+ E. coli strain in comparison with an ung- strain, suggesting that deletions are induced by double-strand breakage of plasmid DNA which occurs in vivo as a result of the excision of opposing uracil residues.

Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: Implications for cancer and neuronal damage

“…** This model is based on studies which show that plasmid DNA containing 2 uracils within 14 base pairs on opposite strands is linearized when transfected into cells expressing uracil-DNA glycosylase (25). Our analysis predicts that the frequency of closely spaced uracil residues on opposite DNA strands increases markedly as uracil accumulates in DNA.…”

“…Therefore, the estimated incidence of 2 closely spaced uracils on opposite strands is 50 times higher in folate-deficient individuals than in normal individuals. Simultaneous removal and strand scission of both closely spaced uracils could cause a double-strand DNA break (25) and would explain the increased levels of micronuclei found in folate-deficient individuals (this time factor is not included in the equation). Since semiconservative DNA replication results in much higher uracil levels in the daughter strands compared with the parental strands, our calculations assume a 1,000-fold difference.…”

“…21), increasing the cellular dUMP͞dTMP ratio and DNA polymerase-mediated dUTP misincorporation into DNA (20,(22)(23)(24). Uracil is excised from DNA by uracil-DNA glycosylase and apyrimidinic endonuclease, generating transient singlestrand breaks (nicks) that could result in a less repairable and more hazardous double-strand break if two opposing nicks are formed (25). Uracil repair-induced double-strand breaks are the likely cause of genetic deletions and duplications in dUTPase mutant Escherichia coli (26).…”

A role for DNA mismatch repair in sensing and responding to fluoropyrimidine damage

“…Additionally, DSBs and deletions could be generated by the repair by UDG of closely spaced uracil residues on opposite strands of DNA (Dianov et al, 1991).…”

Moderate folate depletion modulates the expression of selected genes involved in cell cycle, intracellular signaling and folate uptake in human colonic epithelial cell lines

“…In vitro [15][16][17][18] and in vivo [19][20][21] studies reveal that this nucleotide imbalance promotes the misincorporation of uracil into DNA and the subsequent formation of doublestranded DNA breaks (DSB) through the excision of closely spaced uracil residues on opposing strands during repair [22]. Low dietary folate intake promotes genomic strand breakage in the liver of rats [21,23,24] and micropigs [25].…”