Mismatch Uracil Glycosylase from Escherichia coli

The gene for the mismatch-specific uracil glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the mammalian thymine DNA glycosylase, with activity against uracil in U·G mismatches. Subsequently, 3,N4-ethenocytosine (ϵC), thymine, 5-hydroxymethyluracil, and 8-(hydroxymethyl)-3,N4-ethenocytosine have been proposed as possible substrates for this enzyme. The evaluation of various DNA adducts as substrates is complicated by the biphasic nature of the kinetics of this enzyme. Our results demonstrate that product release by the enzyme is very slow and hence comparing the “steady-state” parameters of the enzyme for different substrates is of limited use. Consequently, the ability of the enzyme to excise a variety of damage products of purines and pyrimidines was studied under single turnover conditions. Although the enzyme excised both ϵC and U from DNA, the former adduct was significantly better as a substrate in terms of binding and hydrolysis. Some products of oxidative and alkylation damage are also moderately good substrates for the enzyme, but thymine is a poor substrate. This comparison of different substrates under single turnover conditions provides a rational basis for comparing substrates of MUG and we relate these conclusions to the known crystal structures of the enzyme and its catalytic mechanism. The gene for the mismatch-specific uracil glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the mammalian thymine DNA glycosylase, with activity against uracil in U·G mismatches. Subsequently, 3,N4-ethenocytosine (ϵC), thymine, 5-hydroxymethyluracil, and 8-(hydroxymethyl)-3,N4-ethenocytosine have been proposed as possible substrates for this enzyme. The evaluation of various DNA adducts as substrates is complicated by the biphasic nature of the kinetics of this enzyme. Our results demonstrate that product release by the enzyme is very slow and hence comparing the “steady-state” parameters of the enzyme for different substrates is of limited use. Consequently, the ability of the enzyme to excise a variety of damage products of purines and pyrimidines was studied under single turnover conditions. Although the enzyme excised both ϵC and U from DNA, the former adduct was significantly better as a substrate in terms of binding and hydrolysis. Some products of oxidative and alkylation damage are also moderately good substrates for the enzyme, but thymine is a poor substrate. This comparison of different substrates under single turnover conditions provides a rational basis for comparing substrates of MUG and we relate these conclusions to the known crystal structures of the enzyme and its catalytic mechanism. DNA glycosylases excise damaged bases from DNA and prevent mutations. They act upon a wide range of DNA adducts that result from the action of a number of DNA damaging agents including water, reactive oxygen species, and alkylating agents. The modified bases that are removed in this way include the products of hydrolytic deamination of cytosine (product: uracil (U)) and 5-methylcytosine (T); the oxidative damage products, 8-oxoguanine and 5-hydroxycytosine (5-OHC) 1The abbreviations used are: OHC, hydroxycytosine; UDG, uracil DNA glycosylase; MUG, mismatch-specific uracil glycosylase; TDG, thymine DNA glycosylase; ϵC, 3,N4-ethenocytosine; HPLC, high performance liquid chromatography; OHU, hydroxyuracil. ; and the alkylation products, 3-methyladenine and 3,N4-ethenocytosine (ϵC). While glycosylases such as uracil DNA glycosylase (UDG) are very specific regarding their substrate (1Savva R. McAuleyhecht K. Brown T. Pearl L. Nature. 1995; 373: 487-493Crossref PubMed Scopus (387) Google Scholar), others, such as endonuclease III, can excise a wide variety of damaged DNA bases (2Thayer M.M. Ahern H. Xing D.X. Cunningham R.P. Tainer J.A. EMBO J. 1995; 14: 4108-4120Crossref PubMed Scopus (437) Google Scholar). The Escherichia coli mismatch-specific uracil glycosylase enzyme (MUG), which was identified as a sequence homolog of human thymine DNA glycosylase (TDG), was so named because it was seen to excise U from a U·G pair (3Gallinari P. Jiricny J. Nature. 1996; 383: 735-738Crossref PubMed Scopus (184) Google Scholar). It was subsequently shown to also excise ϵC and T (4Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8508-8513Crossref PubMed Scopus (164) Google Scholar, 5Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). The crystal structure of MUG revealed that, despite a lack of sequence homology, there was a significant structural homology to UDG (5Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). However, the amino acid residues that confer such a high degree of specificity in UDG were altered in MUG in such a way as to remove any specificity from the active site pocket, which provides a rational basis for its broad substrate specificity (6Barrett T.E. Scharer O.D. Savva R. Brown T. Jiricny J. Verdine G.L. Pearl L.H. EMBO J. 1999; 18: 6599-6609Crossref PubMed Scopus (122) Google Scholar). It appears to gain its specificity from specific interactions with the widowed G in the opposite strand instead (5Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 6Barrett T.E. Scharer O.D. Savva R. Brown T. Jiricny J. Verdine G.L. Pearl L.H. EMBO J. 1999; 18: 6599-6609Crossref PubMed Scopus (122) Google Scholar). Although MUG has been characterized as being active against U, T, and ϵC, it is not yet clear whether these are the true substrates for the enzyme. This uncertainty exists partly because a mug mutant does not have a mutator phenotype in dividing E. coli, and in stationary phase cells, where it is expressed well, it is only a modest mutator (7Lutsenko E. Bhagwat A.S. J. Biol. Chem. 1999; 274: 31034-31038Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Mokkapati S.K. Fernandez de Henestrosa A.R. Bhagwat A.S. Mol. Microbiol. 2001; 41: 1101-1111Crossref PubMed Scopus (23) Google Scholar). It is possible that this lack of a strong phenotype is caused by the presence of alternative enzymes in E. coli that process the promutagenic lesion U and the T·G mispair. Uracils are efficiently removed from DNA by UDG (9Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1974; 9: 3649-3653Crossref Scopus (469) Google Scholar) and T·G mismatches are repaired by a separate repair pathway called very short patch repair (10Lieb M. Bhagwat A.S. Mol. Microbiol. 1996; 20: 467-473Crossref PubMed Scopus (83) Google Scholar, 11Bhagwat A.S. Lieb M. Mol. Microbiol. 2002; 44: 1421-1428Crossref PubMed Scopus (64) Google Scholar). Additionally, there is no evidence, as yet, that E. coli ever generates ϵC in its DNA (12Bartsch H. Nair J. Toxicology. 2000; 153: 105-114Crossref PubMed Scopus (124) Google Scholar). Therefore, it is possible that the biologically important substrate of MUG is something other than U, T, or ϵC. There have been recent reports suggesting that MUG may process a 5-hydroxymethyluracil (13Baker D. Liu P. Burdzy A. Sowers L.C. Chem. Res. Toxicol. 2002; 15: 33-39Crossref PubMed Scopus (40) Google Scholar), 1,N2-ethenoguanine (14Saparbaev M. Langouet S. Privezentzev C.V. Guengerich F.P. Cai H. Elder R.H. Laval J. J. Biol. Chem. 2002; 277: 26987-26993Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and 8-(hydroxymethyl)-3,N4-ethenocytosine (15Hang B. Downing G. Guliaev A.B. Singer B. Biochemistry. 2002; 41: 2158-2165Crossref PubMed Scopus (32) Google Scholar). However, these reports either do not contain quantitative comparisons between relative preferences of the enzyme for different substrates or report apparent steady-state rates of reactions. The latter are problematic, since we demonstrate here that MUG is severely inhibited by its dissociation from abasic product DNA. Hence it is difficult to assess whether these are likely in vivo targets for MUG. If MUG does process a broad diversity of DNA lesions, then it is possible that it acts as a general mismatch DNA glycosylase removing a variety of different lesions that accumulate during the late stationary phase. In this study we have tested this hypothesis by investigating the activity of MUG with a variety of different substrates. We also demonstrate that the use of single turnover kinetics provides a rational means for comparing the rates of nucleotide hydrolysis by MUG. The bases that we have investigated represent different types of damage from a wide variety of possible sources: these include the products of redox reactions, alkylation, and deamination. Our results show that, under the right conditions, MUG can process a broad spectrum of DNA adducts. Protein Purification—The mismatch uracil glycosylase from E. coli (MUG) was overexpressed from a construct of the wild type mug gene in the vector pTrc99A (supplied by Dr R. Savva (Birkbeck, London)) in E. coli strain BL834. An overnight culture of the strain carrying this construct was diluted 1000× into LB broth and grown overnight at 37 °C before induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside. The cultures were grown for a further 5 h at 37 °C before the cells were harvested. The harvested cells were lysed by sonication in buffer A (20 mm Tris-HCl, pH 8.0, 10 mm EDTA) with “Complete” protease inhibitors (Roche Molecular Biochemicals). The lysate was incubated with 1/10 volume of 10% (w/v) streptomycin sulfate for 1 h on ice before centrifugation at 50,000 relative centrifugal force for 1 h to remove the cell debris. The supernatant was loaded onto a DEAE-cellulose column, equilibrated in buffer A, and the flow-through was run directly onto an SP-Sepharose column, also equilibrated in buffer A. The connected columns were washed with 400 ml buffer A; the DEAE column was then removed and the SP Sepharose column washed with a further 150 ml of buffer A. Protein was eluted from the column using a gradient of 0–100% buffer B (20 mm Tris-HCl, pH 8.0, 10 mm EDTA, and 1 m NaCl) over 400 ml. Fractions of the eluate were analyzed by SDS-PAGE and those fractions MUG were and the volume by in a The MUG was loaded onto a column, equilibrated in buffer (20 mm Tris-HCl, pH 8.0, 10 mm EDTA, and m and eluted in the Fractions were as and those MUG were and by The of the was from the on an of was to and of the enzyme were and °C for to and of the damaged tested in this study were into the sequence where is the modified by were using and conditions. were by phase HPLC, as Biochemistry. 1995; PubMed Scopus Google Scholar). substrates were by the modified nucleotide strand with an of the strand either a G or T opposite the damage site which was by and as The were by to °C and to The substrates are to as where is the modified the range of substrates tested and the of the modified bases are shown in by by was by from using the of the was then from this using by by by by by by by by by was by from using the of R. Chem. Res. Toxicol. 1995; PubMed Scopus Google Scholar), the was then from this using by in a substrates were with on the modified strand to The were at °C with MUG in buffer mm Tris-HCl, pH 8.0, mm 1 mm EDTA, and these are the and to other conditions are were removed and with of m The were then to °C for to the abasic site An volume of buffer and mm EDTA) was and subsequently of the were by The of product was using a and the by the to the using 5 MUG a short to were using a The were analyzed as of MUG used a substrate in which strand a single modified DNA modified bases have been investigated as a mismatch with a since the crystal structure of MUG with DNA that the enzyme specific with the opposite the lesion (5Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). However, and lesions with thymine under conditions. Therefore, we have also analyzed the and substrates as mismatches with a thymine the of bases by MUG was seen to it was to conditions under which the enzyme active for so that slow do the enzyme was in the buffer at either or 37 °C for to the of substrate a U·G the of the was to for a of before with It was that this was to of this substrate at either were from to revealed that, at 37 the activity of MUG and h it was no to of the substrate to product the However, at °C enzyme activity was to and a in activity was only h with MUG were at °C and with of to h to that the rates of were as as with the of of MUG under steady-state conditions, were with a of U·G substrate over enzyme for to the there was a in product with The gradient of this steady-state phase a of the of the not the or the but an of This to the enzyme used in the and hence the are of a where the enzyme and an of but where are limited by the very slow release of the steady-state phase of the is not of the true catalytic of the enzyme. a better of the substrate specificity of the enzyme, were under single turnover conditions where the of product a of the that is not by the limited release of with of substrate by MUG was investigated directly by with an of enzyme over substrate. these conditions only a single turnover of the and the of product directly to the of hydrolysis under the conditions were with the and T·G which have been identified as substrates for MUG in (4Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8508-8513Crossref PubMed Scopus (164) Google Scholar, 5Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). The with U·G and than the steady-state and a was to the over of these substrates a single in product with rates that are at of than the steady-state with U·G A and However, the T·G substrate was and h only of the substrate been The single turnover with the U·G substrate that the of this substrate by MUG is than the turnover which limited by the dissociation of product and The product with this enzyme is so that the enzyme is a single turnover enzyme, and it is not possible to the catalytic and for MUG. with MUG were under single turnover conditions. of with single turnover reactions, the of substrate can with enzyme the is not under the conditions whether the rates were to enzyme a of single turnover were with substrates U·G and and of MUG. with the U·G substrate show an in with enzyme which was to a that a of and an apparent of The of the substrate by MUG a very different to the U·G of the rates at were of other and no on the enzyme was It was not possible to the apparent for this which was to from these The is that MUG was with the substrate at the There is a significant in the of MUG for these substrates. by activity of MUG was tested with a of substrates to the possible in vivo targets of the enzyme. The substrates were into with the sequence as the U·G substrate. In the and bases were also opposite a T, which is of conditions and were under single turnover conditions, and the of product was over Some of the substrates tested were and the not to substrate were by the with substrate by comparisons between the rates of MUG with different it is to the under conditions. It was not to do a with these other since the rates were we the with enzyme and 10 which we shown to with the U·G and substrates If the rates not then we that the was and that the was to the catalytic the rates were on enzyme then we that these were not under these conditions of enzyme In the of product a single and to a for the However, the with the T·G and substrates not were so slow that not a In these the were to a and the gradient was used to an The rates of the different substrates at 5 and 10 MUG are shown in the substrates that were at rates under the conditions with the of and The rates of over of and ϵC was the substrate for the enzyme. While a wide variety of modified DNA bases were excised by MUG, the enzyme does not adducts. of the substrates no activity of substrate by the and U·G substrates only the is shown the and U·G substrates only the is shown the and U·G substrates only the is shown in a against is to as specific uracil glycosylase Biochemistry. 2000; PubMed Scopus (40) Google Scholar), and the crystal structure a for the opposite strand in substrate (5Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). the of of the substrates which the enzyme activity were also tested in their single were under the single turnover conditions that were used for the substrates. activity by MUG was only with the substrates that the activity in the was with 10 MUG and ϵC was from single DNA with a of the enzyme to 5 not the of not the enzyme to in a of the not with uracil and not but rates with 5 MUG were and not of this study we have the kinetics of by MUG. conditions under which MUG is active for to h we were to over and of U·G under steady-state conditions revealed a phase by a very slow steady-state phase of the substrate under single turnover conditions that hydrolysis than the turnover biphasic has been with other DNA glycosylases R. J. Mol. Biol. 2002; PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Biochemistry. 1998; PubMed Scopus Google Scholar), and it is clear that the turnover of MUG is also inhibited by binding to its The turnover of MUG is limited by the of product in 1 single turnover conditions, the substrate is with enzyme, the to the of hydrolysis. conditions, the limited by the of the The substrate at enzyme than the U·G substrate This is a of the different binding of the MUG than The binding of the ϵC substrate by MUG also for its activity against DNA ϵC. of the substrate is than the substrate and MUG have a for ϵC in the since it not at the enzyme This the of interactions with the DNA interactions with the are to activity it is in a single DNA However, with the binding the enzyme activity against the single DNA strand was so that it is not likely to of any The activity of MUG in vivo may by the action of other It has been shown that E. coli endonuclease MUG in S.K. Fernandez de Henestrosa A.R. Bhagwat A.S. Mol. Microbiol. 2001; 41: 1101-1111Crossref PubMed Scopus (23) Google Scholar, Biochemistry. 2000; PubMed Scopus (40) Google Scholar), by product the glycosylase is also by endonuclease J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), and the human glycosylases and have been shown to by the human endonuclease P. Jiricny J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Res. 2001; PubMed Scopus Google Scholar). In where the process of is to with abasic product release may not the the activity of MUG with different substrates in it is to single turnover as this a of the relative activity of MUG with different substrates. the different substrates tested a the pair the for the enzyme and was with of While U·G was the of enzyme were to it and was than for the other substrates and rates of than thymine, 5-hydroxymethyluracil, and 5-hydroxycytosine were to and the enzyme not demonstrate any activity and and were the only substrates tested that not under the conditions tested with the biologically T, the a further of It is possible that, in the enzyme may have different substrate act on other sequence or modified by the presence of However, on the results these other to the substrates for the enzyme. have that a variety of other nucleotide lesions are also substrates for MUG, including 5-hydroxymethyluracil (13Baker D. Liu P. Burdzy A. Sowers L.C. Chem. Res. Toxicol. 2002; 15: 33-39Crossref PubMed Scopus (40) Google Scholar), 1,N2-ethenoguanine (14Saparbaev M. Langouet S. Privezentzev C.V. Guengerich F.P. Cai H. Elder R.H. Laval J. J. Biol. Chem. 2002; 277: 26987-26993Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and 8-(hydroxymethyl)-3,N4-ethenocytosine (15Hang B. Downing G. Guliaev A.B. Singer B. Biochemistry. 2002; 41: 2158-2165Crossref PubMed Scopus (32) Google Scholar). However, these reports either do not contain quantitative or report apparent steady-state rates of reactions. The use of steady-state conditions to study MUG and other product inhibited DNA glycosylases to an of rates of the substrates and the substrates better than The for ϵC is only than that for T (4Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8508-8513Crossref PubMed Scopus (164) Google Scholar), the for is the for T·G This is in the that the for ϵC is than the by a of It is difficult from these reports to assess the relative with which MUG is to act upon these different substrates and hence whether are likely in vivo targets for the enzyme. of are that can from the of MUG different substrates. The at the has a on the activity of MUG. This can seen comparing U·G with which has activity and which has of However, is a better substrate than T·G it is It has been that MUG activity is on the of the than P. Burdzy A. Sowers L.C. Chem. Res. Toxicol. 2002; 15: PubMed Scopus Google Scholar). This is with a of with a uracil as proposed for UDG A.R. M. Nature. 2001; PubMed Scopus Google Scholar, J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), and the of R. S. Biochemistry. PubMed Scopus Google Scholar). Our the that there is a between the of any and its on the activity of MUG. In to the poor activity of substrates that have on the and in good substrates. in this is which is the substrate MUG is also to the purines and of ϵC into the active site of MUG has that the active site has to adducts on this of the nucleotide (6Barrett T.E. Scharer O.D. Savva R. Brown T. Jiricny J. Verdine G.L. Pearl L.H. EMBO J. 1999; 18: 6599-6609Crossref PubMed Scopus (122) Google Scholar). The results and (14Saparbaev M. Langouet S. Privezentzev C.V. Guengerich F.P. Cai H. Elder R.H. Laval J. J. Biol. Chem. 2002; 277: 26987-26993Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, B. Downing G. Guliaev A.B. Singer B. Biochemistry. 2002; 41: 2158-2165Crossref PubMed Scopus (32) Google Scholar), demonstrate that these interactions are not specific to ϵC and that adducts can by MUG. It is also of that, with the of other cytosine are poor a comparison of and a of of in the In and cytosine were not substrates for MUG. and A. S. The only structural between these is the presence of a oxygen or amino at the this is to a with the of the single turnover with for the U·G and substrates a in the of the enzyme for these different substrates. there is a in the hydrolysis of these different substrates by MUG, under conditions. The in may to in the of the catalytic residues as the active site to different substrates. these to the of the different In the active site of UDG, an is to specific interactions with the of the uracil (1Savva R. McAuleyhecht K. Brown T. Pearl L. Nature. 1995; 373: 487-493Crossref PubMed Scopus (387) Google Scholar). In MUG, there are no specific interactions to between the of cytosine or the of uracil (6Barrett T.E. Scharer O.D. Savva R. Brown T. Jiricny J. Verdine G.L. Pearl L.H. EMBO J. 1999; 18: 6599-6609Crossref PubMed Scopus (122) Google Scholar). the of the may a a a the This is cytosine is a than hydrolysis of T but hydrolysis of not R. M. Biochemistry. PubMed Scopus (124) Google Scholar). The relative rates of of different substrates by MUG are with the hydrolysis a with a uracil This type of has been to the UDG enzyme A.R. M. Nature. 2001; PubMed Scopus Google Scholar, J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), and it likely that it also to MUG. In this study we have used a to the action of MUG with a broad range of DNA damage We have shown that the use of single turnover kinetics provides a rational basis for comparing the rates of different substrates and to the slow release of MUG from its The substrate for MUG is and it may that this is the biologically substrate. The of MUG that it may active in stationary phase S.K. Fernandez de Henestrosa A.R. Bhagwat A.S. Mol. Microbiol. 2001; 41: 1101-1111Crossref PubMed Scopus (23) Google Scholar), and hence it is possible that E. coli DNA ϵC during this of cell

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