A major threat to the genetic integrity of living cells comes from endogenous DNA damage, due to the unavoidable exposure of DNA to reactive molecules such as active oxygen and water during normal cellular metabolism. One of the most abundant miscoding DNA lesions is uracil, which can arise in DNA by hydrolytic deamination of cytosine, generating a premutagenic U:G mispair and C to T transition mutations. Uracil may also occur in a U:A base pair through occasional use of dUTP instead of TTP during DNA replication. The repair of uracil in DNA is achieved by means of the base-excision repair pathway, and initiated via excision of uracil by a DNA glycosylase. Multiple uracil-DNA glycosylase enzymes are widely distributed in bacteria, archaea and eukaryotes, signifying the importance of eliminating uracil from the genome. We have previously reported the generation of gene-targeted mice deficient in the ubiquitous Ung uracil-DNA glycosylase. A primary role for Ung during DNA replication was revealed, where slow removal of uracil from misincorporated dUMP resulted in a ∼100-fold increased steady-state level of uracil in the genome of Ung-/- mice. This is consistent with the proliferation-dependent expression of UNG in the mammalian cell nucleus, its direct interaction with PCNA in the replisome, and a major role of Ung in counteracting U:A base pairs. In collaboration with the laboratory of Michael Neuberger (MRC Laboratory of Molecular Biology, Cambridge, UK), Ung was also shown to have a role in resolving U:G lesions, specifically generated by the AID enzyme (activation induced cytidine deaminase) in immunoglobulin variable genes during somatic hypermutation and class switch recombination. In support of this function for Ung in the immune system, we have reported that aging Ung-/- null mice have a greatly increased incidence of B-cell lymphomas. The molecular mechanism of lymphomagenesis remains unclear, although it is in some cases correlated with deregulation of the AID enzyme in Ung-/- tumours; it has yet to be established whether patients with hyper-IgM syndrome due to mutations in the human UNG gene are at an elevated risk of B-cell malignancies. A complementary uracil-excising activity was identified in Ung-deficient mice that acts on premutagenic U:G lesions resulting from genome-wide hydrolytic deamination of cytosine. This second major uracil-DNA glycosylase, which has evolved in insects and vertebrates, is due to the SMUG enzyme, and accounts for the only modest mutator phenotype in Ung-deficient mammalian cells, in contrast to the greatly increased frequency of spontaneous C to T mutations in Ung null mutants of bacteria and yeast. We have now generated stable siRNA-mediated Smug knockdown cell lines and transgenic mice, on both an Ung-proficient and -deficient background, to address: (i) the role of SMUG as an anti-mutator, and (ii) the delineation/overlap of function between the UNG and SMUG uracil-DNA glycosylases, in maintenance of the mammalian genome.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]