Mammalian 5-Formyluracil−DNA Glycosylase. 2. Role of SMUG1 Uracil−DNA Glycosylase in Repair of 5-Formyluracil and Other Oxidized and Deaminated Base Lesions

Abstract

In the accompanying paper [Matsubara, M., et al. (2003) Biochemistry 42, 4993-5002], we have partially purified and characterized rat 5-formyluracil (fU)-DNA glycosylase (FDG). Several lines of evidence have indicated that FDG is a rat homologue of single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1). We report here that rat and human SMUG1 (rSMUG1 and hSMUG1) expressed from the corresponding cDNAs indeed excise fU in single-stranded (ss) and double-stranded (ds) DNA. The enzymes also excised uracil (U) and uracil derivatives bearing an oxidized group at C5 [5-hydroxyuracil (hoU) and 5-hydroxymethyluracil (hmU)] in ssDNA and dsDNA but not analogous cytosine derivatives (5-hydroxycytosine and 5-formylcytosine) and other oxidized damage. The damage specificity and the salt concentration dependence of rSMUG1 (and hSMUG1) agreed well with those of FDG, confirming that FDG is rSMUG1. Consistent with the damage specificity above, hSMUG1 removed damaged bases from Fenton-oxidized calf thymus DNA, generating abasic sites. The amount of resulting abasic sites was about 10% of that generated by endonuclease III or 8-oxoguanine glycosylase in the same substrate. The HeLa cell extract and hSMUG1 exhibited a similar damage preference (hoU.G > hmU.A, fU.A), and the activities for fU, hmU, and hoU in the cell extract were effectively neutralized with hSMUG1 antibodies. These data indicate a dual role of hSMUG1 as a backup enzyme for UNG and a primary repair enzyme for a subset of oxidized pyrimidines such as fU, hmU, and hoU.

Thymine DNA Glycosylase Can Rapidly Excise 5-Formylcytosine and 5-Carboxylcytosine

“…Regarding a potential role for other glycosylases in the excision of fC and caC, it has been shown that SMUG1 cannot remove fC from DNA (46). We find that MBD4 (catalytic domain) has ϳ120-fold lower activity for G⅐fC relative to a G⅐T substrate and that G⅐fC activity is ϳ450-fold lower for MBD4 as compared with TDG.…”

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

“…The structure of human UDG and that bound to uracil-containing oligo has demonstrated the basis for the enzyme-assisted nucleotide flipping [21,22]. Another DNA glycosylase SMUG1 has been characterized 30 npg in human cells [23,24], which catalyzes excision of U and also oxidized pyrimidines such as 5-hydroxycytosine (5-OHC). SMUG1 is a member of UDG family of glycosylases.…”

Base Excision Repair and Lesion-Dependent Subpathways for Repair of Oxidative DNA Damage

“…Although it has been suggested that hmdC may be removed by a mammalian DNA glycosylase (12), the identity of this glycosylase is unknown. However, deamination of hmdC yields 5-hydroxymethyl-uracil (hmdU), a substrate for SMUG1, TDG, and MBD4 (64), suggesting that the hmdC lesion may be cleared from genomic DNA by deamination followed by BER. In addition to lesions formed by direct oxidative damage to DNA from ROS, chronic induction of ROS promulgates a host of cellular alterations, including significantly elevated levels of LPO (5).…”