Presence and consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats

Uracil can arise in DNA by misincorporation of dUTP into nascent DNA and/orby cytosine deamination in established DNA. Based on recent findings, bothpathways appear to be promoted in the methyl-deficient model ofhepatocarcinogenesis. A chronic increase in the ratio dUTP:dTTP withfolate/methyl deficiency can result in a futile cycle of excision andreiterative uracil misincorporation leading to premutagenic apyrimidinic(AP) sites, DNA strand breaks, DNA fragmentation and apoptotic cell death.The progressive accumulation of unmethylated cytosines with chronic methyldeficiency will increase the potential for cytosine deamination to uraciland further stress uracil mismatch repair mechanisms. Uracil is removed bya highly specific uracil-DNA glycosylase (UDG) leaving an AP site that issubsequently repaired by sequential action of AP endonuclease,5'-phosphodiesterase, a DNA polymerase and DNA ligase. Since the DNApolymerases cannot distinguish between dUTP and dTTP, an increase indUTP:dTTP ratio will promote uracil misincorporation during both DNAreplication and repair synthesis. The misincorporation of uracil forthymine (5-methyluracil) may constitute a genetically significant form ofDNA hypomethylation distinct from cytosine hypomethylation. In the presentstudy a significant increase in the level of uracil in liver DNA as earlyas 3 weeks after initiation of folate/methyl deficiency was accompanied byparallel increases in DNA strand breaks, AP sites and increased levels ofAP endonuclease mRNA. In addition, uracil was also detected within the p53gene sequence using UDG PCR techniques. Increased levels of uracil in DNAimplies that the capacity for uracil base excision repair is exceeded withchronic folate/methyl deficiency. It is possible that enzyme-inducedextrahelical bases, AP sites and DNA strand breaks interact to negativelyaffect the stability of the DNA helix and stress the structural limits ofpermissible uracil base excision repair activity. Thus substitution ofuracil for thymine induces repair- related premutagenic lesions and a novelform of DNA hypomethylation that may relate to tumor promotion in themethyl-deficient model of hepatocarcinogenesis.