Efficient removal of uracil from G.U mispairs by themismatch-specific thymine DNA glycosylase from HeLa cells.

Abstract

The uracil DNA glycosylases (EC 3.2.2.3) characterized to date remove uracil from DNA irrespective of whether it is base paired with adenine or mispaired with guanine in double-stranded substrates or whether it is found in single-stranded DNA. We report here the characterization of uracil glycosylase activity that can remove the base solely from a mispair with guanne. It does not recognize uracil either in A-U pairs or in single-stranded substrates. The enzyme, a 55-kDa polypeptide, was previously characterized as a mismatch-specific thymine DNA glycosylase and was thought to be responsible solely for the correction (to GaC) of G T mispais that arise as a result of spontaneous hydrolytic deaination of 5-methylcytosine to thymine. Given the broader substrate specificity of the enzyme (in addition to uracil and thymine, the protein can also remove 5-bromouracil from mispairs with guanine), we propose that its biological role in vivo may also incinde the correction of a subset of GNU mips inefficiently removed by the more abundant ubiquitous uraci glycosylases, such as those arising from cytosine deatnation in G+C-rich regions of the genome.Uracil residues arise in DNA either by cytosine deamination (1) or by incorporation into the nascent strand during replication as dUMP (2). The former process gives rise to mutagenic G-U mispairs, whereas the latter yields A-U "pairs." Uracil is removed from both these lesions and from singlestranded DNA by ubiquitous uracil DNA glycosylases (EC 3.2.2.3) (2, 3). A recent study addressing the removal ofuracil from a large number ofdifferent sequence contexts suggested that the major uracil glycosylases of both mammals and bacteria may require local strand separation for efficient removal of the base from DNA (4). Correspondingly, G-U mispairs in a G+C-rich sequence context were found to be poor substrates for these enzymes in vitro, although this may not be so for all uracil glycosylases (5). Interestingly, analysis of mutational spectra of several mammalian genes published to date revealed no evidence for an increased rate of C --T transition mutations that could be associated with G+C-rich regions (6, 7). This would imply either that the ubiquitous enzyme is so abundant that it manages to repair even the less accessible substrates or that a second enzyme may be able to compensate for the inefficiency ofthe ubiquitous activity. We describe here another glycosylase activity that, due to its different substrate recognition requirements, efficiently removes uracil from GNU mispairs in double-stranded DNA and may thus be a candidate for such a back-up role. MATERIALS AND METHODSAll the reagents and solvents used in this study were of analytical purity. The Preparation of Whole Cell Extracts. These extracts were prepared from 180 g offrozen HeLa cells in three 60-g batches as described (9).Fractionation of the Cell Extracts. The extract was diluted 1:4 with HE buffer [25 mM Hepes, pH 7.8/1 mM EDTA/1 mM dithiothreitol/10% (vol/vol) glycerol] and incubated batchwise for 1 h w...

Biphasic Kinetics of the Human DNA Repair Protein MED1 (MBD4), a Mismatch-specific DNA N-Glycosylase

“…MED1 Is Also Active on Uracil and 5-Fluorouracil Paired with Guanine-Both Mig.Mth and TDG have a mismatch-specific uracil glycosylase activity (22,31). Based on the similarities with these enzymes, we tested the uracil glycosylase activity of MED1 on oligonucleotide substrates in which uracil was paired with A, C, G, or T. As expected, MED1 uracil glycosylase activity is specific for G:U mismatches (Fig.…”

Fidelity of Uracil-initiated Base Excision DNA Repair in DNA Polymerase β-Proficient and -Deficient Mouse Embryonic Fibroblast Cell Extracts

“…The observation that Ugi inactivates these biologically distinct enzymes was not surprising because these proteins share 55.7% identical amino acid residues (23,26). In contrast, TDG, MED1, and SMUG, which do not share similar amino acid homology with UNG, are insensitive to inhibition by Ugi (13,18,27,28). Thus, addition of Ugi to cell-free extracts has been effectively used to investigate Ugisensitive and Ugi-insensitive modes of uracil-initiated BER (21,29).…”

“…UNG preferentially recognizes uracil residues in single-stranded DNA and is also active on duplex DNA containing U⅐G mispairs and U⅐A base pairs (17). In contrast, human thymine-DNA glycosylase does not recognize uracil in single-stranded DNA but does excise uracil residues in double-stranded DNA with the following specificity: U⅐G Ͼ U⅐C Ͼ U⅐T Ͼ Ͼ U⅐A (18,19). The human MED1 (also termed MBD4) enzyme acts on U⅐G mispairs in the context of methylated or unmethylated CpG sites, but activity against uracil-containing single-stranded DNA and U⅐A, U⅐C, or U⅐T double-stranded DNA substrates was not detected (20).…”

Repair of exocyclic DNA adducts: rings of complexity

“…(51) Through an extensive purification, the eC-DNA glycosylase was identified as a 55 kDa polypeptide by SDS-PAGE, (52) which is the exact molecular mass of the previously purified human mismatchspecific T(U) Á G-DNA glycosylase, termed thymine-DNA glycosylase (TDG). (53) Moreover, the T Á G and U Á G mismatch glycosylase activities co-eluted with the eC activity in the same fractions, and competition studies suggested that they all reside in the same protein. (52) It was then proposed that eC is a substrate for hTDG.…”