Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20؋ the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein.

BackgroundRNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms. We performed a small interfering (si)RNA screen for novel members of the transforming growth factor (TGF)-β pathway in a human keratinocyte cell line. The TGF-β pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-β responses have been strongly implicated in cancer.ResultsWe assayed how strongly single siRNAs targeting each of 6,000 genes affect the nuclear translocation of a green fluorescent protein (GFP)-SMAD2 reporter fusion protein. Surprisingly, we found no novel TGF-β pathway members, but we did find dominant off-target effects. All siRNA hits, whatever their intended direct target, reduced the mRNA levels of two known upstream pathway components, the TGF-β receptors 1 and 2 (TGFBR1 and TGFBR2), via micro (mi)RNA-like off-target effects. The scale of these off-target effects was remarkable, with at least 1% of the sequences in the unbiased siRNA library having measurable off-target effects on one of these two genes. It seems that relatively minor reductions of message levels via off-target effects can have dominant effects on an assay, if the pathway output is very dose-sensitive to levels of particular pathway components. In search of mechanistic details, we identified multiple miRNA-like sequence characteristics that correlated with the off-target effects. Based on these results, we identified miR-20a, miR-34a and miR-373 as miRNAs that inhibit TGFBR2 expression.ConclusionsOur findings point to potential improvements for miRNA/siRNA target prediction methods, and suggest that the type II TGF-β receptor is regulated by multiple miRNAs. We also conclude that the risk of obtaining misleading results in siRNA screens using large libraries with single-assay readout is substantial. Control and rescue experiments are essential in the interpretation of such screens, and improvements to the methods to reduce or predict RNAi off-target effects would be beneficial.

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Base excision repair of oxidized pyrimidines in human DNA is initiated by the DNA N-glycosylase/ apurinic/apyrimidinic (AP) lyase, human NTH1 (hNTH1), the homolog of Escherichia coli endonuclease III (Nth). In contrast to Nth, the DNA N-glycosylase activity of hNTH1 is 7-fold greater than its AP lyase activity when the DNA substrate contains a thymine glycol (Tg) opposite adenine (Tg: Like its Escherichia coli homolog, endonuclease III (Nth), hNTH1 1 is a bifunctional DNA N-glycosylase/apurinic/apyrimidinic (AP) lyase that removes ring-saturated pyrimidines, be they hydrated, reduced, or oxidized, from the DNA backbone as the initial step of base excision repair (BER) of such modified residues (1). The oxidation product of thymine, 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol (Tg)) is a widely studied model substrate for hNTH1 and catalyzes its release from DNA via its DNA N-glycosylase activity. This enzyme activity is mediated via formation of a transient Schiff base (imino) enzyme-DNA intermediate, an example of covalent catalysis and a characteristic of all known bifunctional DNA N-glycosylases/AP lyases (4, 5). The Schiff base moiety has been hypothesized to be required for the enzymatic catalysis of the ␤-elimination reaction, which effects DNA strand cleavage 3Ј to the abasic (AP) site formed as a product of the release of the base from the 2Ј-deoxyribose moiety to which it was linked. The hydrolysis of the Schiff base intermediate can occur in the absence of or following enzyme-catalyzed ␤-elimination (AP lyase activity), resulting in DNA strand cleavage. The enzymatic catalysis of ␤-elimination by DNA N-glycosylases/AP lyases has been shown to be initiated via abstraction of the deoxyribose pro-S-2Ј-hydrogen by a basic amino acid in the enzyme active site. Several factors, including pH, can affect the efficiency of the AP lyase step by affecting substrate binding and proton abstraction (5).ABased on the results of studies with Nth, the two activities of hNTH1 (DNA N-glycosylase and ␤-elimination catalysis) require the formation of the Schiff base intermediate and were thought to occur concomitantly. However, data from our laboratory (1) and from other laboratories (6 -8) indicate that DNA N-glycosylase and ␤-elimination catalysis by mammalian members of the endonuclease III enzyme superfamily are not concurrent under the assay conditions employed. We were the first to report that the DNA N-glycosylase and AP lyase activities of hNTH1 are not concurrent, i.e. that the rate of AP lyase-mediated strand cleavage is much slower than the rate of DNA N-glycosylase-mediated base release. These results are similar to the non-concurrence of base release and strand cleavage reported for the mammalian 8-oxoguanine-DNA N-glycosylase homolog OGG1 (8). In this report, we present data demonstrating that the dissociation of the two activities of hNTH1 is dependent on the nature of the orphan base opposite the Tg residue in DNA.In our studies of the properties of hNTH1, we have focused

DNA N-glycosylase/AP (apurinic/apyrimidinic) lyase enzymes of the endonuclease III family (nth in Escherichia coli and Nth1 in mammalian organisms) initiate DNA base excision repair of oxidized ring saturated pyrimidine residues. We generated a null mouse (mNth1 ؊/؊ ) by gene targeting. After almost 2 years, such mice exhibited no overt abnormalities. Tissues of mNth1 ؊/؊ mice contained an enzymatic activity which cleaved DNA at sites of oxidized thymine residues (thymine glycol [Tg]). The activity was greater when Tg was paired with G than with A. This is in contrast to Nth1, which is more active against Tg:A pairs than Tg:G pairs. We suggest that there is a back-up mammalian repair activity which attacks Tg:G pairs with much greater efficiency than Tg:A pairs. The significance of this activity may relate to repair of oxidized 5-methyl cytosine residues (5meCyt). It was shown previously (S. Zuo, R. J. Boorstein, and G. W. Teebor, Nucleic Acids Res. 23:3239-3243, 1995) that both ionizing radiation and chemical oxidation yielded Tg from 5meCyt residues in DNA. Thus, this previously undescribed, and hence novel, back-up enzyme activity may function to repair oxidized 5meCyt residues in DNA while also being sufficient to compensate for the loss of Nth1 in the mutant mice, thereby explaining the noninformative phenotype.Escherichia coli endonuclease III and its eukaryotic homologs initiate the process of DNA base excision repair (BER) of C-5, C-6 ring saturated pyrimidines (31). The endonuclease III-like enzymes are bifunctional enzymes which first effect release of the damaged base from the DNA backbone and then effect strand cleavage at the resulting abasic (apurinic/apyrimidinic [AP]) site via ␤-elimination (19,20,32). Among the modified bases recognized by endonuclease III-like enzymes are 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol [Tg]), cytosine glycol, and cytosine hydrate. Tg is an oxidative product formed in DNA in vitro by reaction with chemical oxidizing agents such as KMnO 4 and OsO 4 as well as via the indirect action of ionizing radiation under aerobic conditions. In cellular DNA, Tg is formed as a product of exposure to aerobic ionizing radiation and other oxidative stresses such as H 2 O 2 in the presence of added Fe 2ϩ . Ionizing irradiation under anaerobic conditions yields the reduced derivative 5,6-dihydrothymine (4, 9, 28). Exposure of cytosine to oxidative stress yields cytosine glycol, which is in equilibrium with its dehydrated form, 5-hydroxycytosine (5ohCyt). 5ohCyt is prone to deamination yielding 5ohUra. In addition to inducing formation of dimeric photoproducts in DNA, UV irradiation also induces hydration of the 5,6 double bond of pyrimidines, primarily forming 5,6 dihydro-6-hydroxy cytosine (cytosine hydrate) (2, 3). Like 5ohCyt, cytosine hydrate is prone to deamination, which yields uracil hydrate (26).Our laboratory first documented the existence of a family of endonuclease III homologs in several species (11) and isolated the cDNA of the human homolog of endonuclease III (hNth...