Kinetics and binding of the thymine-DNA mismatch glycosylase, Mig-Mth, with mismatch-containing DNA substrates

DNA Repair 2 (2003) 107–120

Kinetics and binding of the thymine-DNA mismatch glycosylase, Mig-Mth, with mismatch-containing DNA substrates Thomas J. Begley a,1 , Brian J. Haas a , Juan C. Morales b , Eric T. Kool b , Richard P. Cunningham a,∗ a

Department of Biological Sciences, SUNY at Albany, Albany, NY 12222, USA b Department of Chemistry, Stanford University, Stanford, CA 94305, USA Accepted 26 September 2002

Abstract We have examined the removal of thymine residues from T–G mismatches in DNA by the thymine-DNA mismatch glycosylase from Methanobacterium thermoautrophicum (Mig-Mth), within the context of the base excision repair (BER) pathway, to investigate why this glycosylase has such low activity in vitro. Using single-turnover kinetics and steady-state kinetics, we calculated the catalytic and product dissociation rate constants for Mig-Mth, and determined that Mig-Mth is inhibited by product apyrimidinic (AP) sites in DNA. Electrophoretic mobility shift assays (EMSA) provide evidence that the specificity of product binding is dependent upon the base opposite the AP site. The binding of Mig-Mth to DNA containing the non-cleavable substrate analogue difluorotoluene (F) was also analyzed to determine the effect of the opposite base on Mig-Mth binding specificity for substrate-like duplex DNA. The results of these experiments support the idea that opposite strand interactions play roles in determining substrate specificity. Endonuclease IV, which cleaves AP sites in the next step of the BER pathway, was used to analyze the effect of product removal on the overall rate of thymine hydrolysis by Mig-Mth. Our results support the hypothesis that endonuclease IV increases the apparent activity of Mig-Mth significantly under steady-state conditions by preventing reassociation of enzyme to product. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Base excision repair; DNA glycosylase; Endonuclease III; TDG; Mig

1. Introduction Abbreviations: AP site, apyrimidinic/apurinic site; BER, base excision repair; CHES, 2-(cyclohexylamino)-ethanesulphonic acid; EMSA, electrophoretic mobility shift assays; F, difluorotoluene; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid; kcat-st , single-turnover rate constant; k3 , product dissociation rate constant; kcat , catalytic rate constant; Kd , substrate dissociation constant; MBD, methyl binding domain; Mig, mismatch DNA glycosylase; UDG, uracil-DNA glycosylase ∗ Corresponding author. Tel.: +1-518-442-4331; fax: +1-518-442-4767. E-mail address: [email protected] (R.P. Cunningham). 1 Present address: Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

DNA glycosylases initiate DNA base excision repair by removing an inappropriate or damaged base from DNA [1–4]. Mono-functional DNA glycosylases include UDG, TDG, MutY, mismatch DNA glycosylase from Methanobacterium thermoautrophicum (Mig-Mth) MBD4 (MED1), and the alkylpurine glycosylases [2,5–8]. These enzymes differ in substrate specificity, but all generate a common product, an intact AP site. The repair of these AP sites involves processing by an AP endonuclease that cleaves the

1568-7864/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 8 - 7 8 6 4 ( 0 2 ) 0 0 1 9 0 - 8

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phosphodiester backbone 5 to the AP site. Deoxyribophosphodiesterase then removes the resulting deoxyribose-5 -phosphate, to create a single-nucleotide gap in the DNA, which is filled and sealed by the successive action of DNA polymerase and DNA ligase [9,10]. In mammalian cells, a second base excision repair pathway creates and fills gaps of 2–13 nucleotides. Termed long-patch repair, this pathway utilizes an AP endonuclease, FEN I, PCNA, ␤-pol, XRCC1, and DNA ligase III to repair AP sites [10]. Mig-Mth recognizes T–G and U–G mismatches and removes the pyrimidine opposite the guanine [11,12]. These mismatches arise from deamination events that convert cytosine into uracil and the minor base 5-methylcytosine (5meC) into thymine [13]. Failure to remove these deamination products leaves mismatched U–G and T–G base pairs in DNA that result in C–G to T–A transition mutations upon replication [14]. The gene encoding Mig-Mth was found on a plasmid, pFV1, that also encodes a modification system that methylates at the C5 position of the internal cytosine in a CCGG sequence [15]. This is an unexpected modification system for a thermophilic organism because of the high rate of deamination of 5meC at 65 ◦ C [16]. Mig-Mth, as part of a base excision repair pathway, has been implicated in counteracting the mutagenic effects of the spontaneous deamination of 5meC residues [11,17]. We have also identified a potential Mig gene in the Aeropyrum pernix genome that is near a potential 5-methylcytosine-DNA methyltransferase. A Mig from Pyrobaculum aerophilum has also been described [18]. In addition, the human enzyme MBD4 (MED1), contains a catalytic domain similar in sequence to the Mig’s [7,8] suggesting that there is a family of these enzymes in nature. These Mig enzymes represent one of two distinct classes of thymine-DNA mismatch glycosylases. Members of the other class are human TDG and the Escherichia coli mismatch specific uracil glycosylase (MUG). These enzymes are functional analogs of Mig, but show no similarity to the primary amino acid sequence of Mig [19]. AP sites can be introduced into DNA both by glycosylases and by spontaneous depurination and depyrimidination events. AP sites represent a generic form of damage that is the common product of a number of different pure glycosylases and of spontaneous depurination and depyrimidination events. The efficient

processing of these sites is extremely important because DNA containing AP sites is non-instructive during replication. Unrepaired AP sites can lead to the arrest of the replication fork and cell death [20]. Imbalanced base excision repair, resulting in the accumulation of AP sites, has also been linked to increased mutation rates [20–23]. AP endonucleases initiate the repair of AP sites. There are two families of AP endonucleases; the prototypical enzymes of these families are exonuclease III and endonuclease IV of E. coli [24,25]. Exonuclease III homologs have been found in bacteria, archaea and in eukaryotes including Drosophilia, Arabadopsis and Homo sapiens. Endonuclease IV homologs have been identified in bacteria, archaea and lower eukaryotes including baker’s yeast [26]. We report here on studies carried out to investigate some of the factors affecting Mig-Mth activity in vitro. We have measured Mig-Mth activity using single-turnover kinetics and steady-state kinetics. A thermophilic AP endonuclease was used to reconstitute the initial steps of the base excision repair pathway. Kinetic analysis of Mig-Mth in the presence of endonuclease IV was employed to determine the effect of product removal on the glycosylase activity. Gel retardation experiments using oligonucleotides containing AP sites or difluorotoluene, a non-cleavable substrate analogue of thymine, were used to analyze the binding of Mig-Mth to substrate and product and to evaluate the importance of the base opposite these lesions. The results of these experiments support the hypothesis that endonuclease IV increases the apparent activity of Mig-Mth by preventing reassociation of enzyme to product and that opposite strand interactions play roles in determining substrate specificity and product affinity. 2. Materials and methods 2.1. Enzymes Mig-Mth was purified as previously described [12]. Thermotoga maritimama endonuclease IV was purified to apparent homogeneity as previously described [27]. The concentration of Mig-Mth was determined spectroscopically using a millimolar extinction coefficient of 14.4 mM−1 cm−1 at 410 nm as previously

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described [12]. The concentration of endonuclease IV was determined using a millimolar extinction coefficient of 30.6 mM−1 cm−1 at 280 nm. 2.2. Substrates The convention we use to designate DNA duplexes consists of letters to identify the type of mismatch or product created from the annealing of 19 bp-long oligonucleotides. The oligonucleotides were complementary except at a central position creating either a mismatch or an AP site upon annealing. The strand that was radioactively labeled is listed first. Oligonucleotides T (5 -GCAGCGCAGTCAGCCGACG-3 ), U (5 -GCAGCGCAGUCAGCCGACG-3 ), and G (5 -CGTCGGCTGGGCCCTGCGCTGC-3 ) were purchased from Oligos Etc. and were supplied as lyophilized samples, which were used without further purification. Oligonucleotides A (5 -CGTCGGCTGGACCCTGCGCTGC-3 ), T2 (5 CGTCGGCTGGTCCCTGCGCTGC-3 ), and C (5 -CGTCGGCTGGCCCCTGCGCTGC-3 ) were synthesized on an ABI 392 synthesizer. These oligonucleotides were purified using the Oligo Prep kit and the Easy Prep system (Pharmacia Biotech Inc.). The difluorotoluene-containing oligonucleotide, F (5 -GCAGCGCAGFCAGCCGACG-3 ), was prepared as previously described [28,29]. Duplex DNA molecules were prepared as previously described [12]. Briefly, single-stranded oligonucleotides were labeled with ␥-[32 P] ATP (New England Nuclear) using T4 polynucleotide kinase (New England BioLabs). Unincorporated ATP was removed using Sephadex G25 spin columns (Pharmacia Biotech Inc.). The oligonucleotides were annealed at a 1:1 ratio for 1 h to form stable duplexes. Oligonucleotides containing AP sites were prepared as previously described [12]. Briefly, the uracil in oligonucleotide U was removed by treatment for 1 h at 37 ◦ C in 70 mM HEPES–NaOH pH 7.5, 10 mM MgCl2 , 0.1 mM DTT with 50 ng E. coli uracil glycosylase to make an AP 19-mer. Oligonucleotides G, A, C and T2 were annealed with this AP 19-mer to make product oligonucleotides AP–G, AP–A, AP–C, and AP–T2. 2.3. Glycosylase assays The activity of Mig-Mth was monitored by measuring the alkali-induced cleavage of AP sites cre-

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ated by the enzyme acting on a mismatched base pair. Indicated amounts of a mismatched [32 P] labeled substrate and enzyme were incubated in 50 mM HEPES–KOH pH 7.5, 100 mM NaCl at 65 ◦ C for specified times. The enzyme was inactivated and the AP sites cleaved by the addition of 0.15 M NaOH followed by incubation at 65 ◦ C for 15 min. Loading buffer (80% deionized formamide, 50 mM HEPES–KOH pH 7.5, 1 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol) was added to the samples, which were then run on a 20% polyacrylamide gel (8.0 cm × 7.3 cm × 0.75 cm) containing 8 M urea in 50 mM HEPES–NaOH pH 7.5 for 2 h at 200 V. Gels were exposed to a Molecular Dynamics phosphorimager screen and the phosphor autoradiogram was quantified on a Storm phosphorimager. 2.4. Single-turnover kinetics To measure single-turnover kinetics of thymine removal by Mig-Mth, we monitored product formation under conditions where the enzyme was in excess of substrate; 47 ␮M Mig-Mth was incubated with 15 ␮M oligonucleotide containing a central T–G mismatch. The addition of NaOH at 0, 0.5, 1, 2, 3, 4, 6, or 8 min terminated the reaction. The zero-time point in the presence of Mig-Mth measures the amount of product formed before Mig-Mth could be inactivated. Similarly, single-turnover kinetics of thymine hydrolysis was studied using 47 ␮M Mig-Mth and endonuclease IV, at concentrations listed in Table 1, with 15 ␮M oligonucleotide containing a T–G mismatch. The products of these reactions were run on denaturing gels as described above. The gels were analyzed as described above. Experiments were performed in triplicate.

Table 1 Mig-Mth kinetic constants determined under single-turnover conditions Additions

kcat-st (min−1 )a

None 1.3 ␮M endonuclease IV 90 ␮M endonuclease IV

0.68 ± 0.069 0.76 ± 0.068 0.80 ± 0.11

a

[Mig-Mth] for single-turnover kinetic experiments: 47 ␮M.

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2.5. Time course assay Thymine mismatch glycosylase activity was monitored over a 2 h time course using 40 ␮M T–G substrate, 5 ␮M Mig-Mth in either the absence or presence of 1.3 ␮M endonuclease IV. The reactions were performed as described above, and were terminated by the addition of NaOH. The amount of product produced was determined and plotted for time points between 0 and 105 min. Experiments were performed in triplicate. 2.6. Gel retardation experiments Each reaction contained 47 nM oligonucleotides containing an AP or F strand annealed to a G, A, C or T2 strand, 0 to 32 ␮M Mig-Mth, 50 mM CHES–KOH pH 9.2, 100 mM KCl, 10 mM EDTA, 15% glycerol, and nuclease-free BSA (1 ␮g/␮l). Reaction mixtures were incubated at room temperature for 30 min prior to loading on a 8% polyacrylamide (19:1) gel (8.0 cm × 7.3 cm × 1.0 cm) containing 50 mM CHES–KOH pH 9.2. The gels were run at 150 V in 50 mM CHES–KOH pH 9.2 for 35 min at room temperature. The radioactivity of free and complexed DNA was quantified on a Storm phosphorimager. Experiments were performed in triplicate.

(Fig. 1B). The use of enzyme and substrate at high concentrations assumes that the enzyme–substrate complex forms rapidly and does not effect the rate equation, allowing these experiments to be analyzed by simple chemical kinetics. Experiments designed to measure the rate constant for enzyme binding to substrate showed that this process is to fast to measure manually (unpublished observations), thus our assumptions seem warranted. 3.2. Time course of Mig-Mth action Our previous results [12] showed that Mig-Mth binds to product and substrate with similar affinities. This observation would predict that product would serve as an inhibitor of enzyme activity and, thus, that the time course of enzyme action should not be linear. We therefore monitored the extent of product formation as a function of time under multiple-turnover conditions (Fig. 2A). Using Mig-Mth alone, we observed an initial burst of product formation followed by a period of decreasing rate of product formation (Fig. 2B, closed circles). There was no linear phase of steady-state product formation, as we had anticipated based on the tight binding of enzyme to product. We were able to fit the initial burst phase to the following equation: [product] = A(1 − exp{(−kcat )(time)})

3. Results 3.1. Single-turnover kinetics Our previous results [12] suggested that tight product binding would make Michaelis–Menten kinetic analysis difficult for Mig-Mth, so we performed single-turnover experiments to directly measure the single-turnover rate constant kcat-st . We conducted these experiments under conditions with enzyme in excess of substrate and with substrate concentrations above Kd . The formation of product AP sites followed first order kinetics (Fig. 1A), and we determined the single-turnover catalytic rate constant, kcat-st , using the following equation: [product] = A(1 − exp{(−kcat-st )(time)})

where A is a normalization factor representing the total active enzyme concentration and kcat is the catalytic rate constant. We obtained a value of 0.83 ± 0.10 min−1 that is in good agreement with the value that we obtained under single-turnover conditions (Table 1). Once the initial burst is over, enzyme can bind either to substrate or product, and the rate of the reaction should decrease as product accumulates. The reaction does not approach completion even after 105 min (Fig. 2A, top). A simple reaction scheme consistent with this data and data presented in following sections is shown in Scheme 1. If product serves as a competitive inhibitor of the enzyme, we should be able to monitor uninhibited steady-state kinetics

(1)

where A represents the amplitude of the exponential phase. We obtained a value of 0.68 ± 0.06 min−1

(2)

Scheme 1. Kinetic scheme for Mig-Mth (kcat = kcat-st ).

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Fig. 1. (A) Storage phosphor autoradiogram of a denaturing polyacrylamide gel containing samples in which Mig-Mth and an oligonucleotide containing a T–G mismatch were incubated at 65 ◦ C for various times up to 8 min. After incubation, the samples were treated with base to stop the reaction and cleave the phosphodiester backbone at AP sites. Lane 2 shows the amount of product formation that occurs before the reaction can be quenched. (B) Single-turnover kinetics of Mig-Mth plotted from the data in (A) and fitted to Eq. (1). The concentration of product is plotted as a function of time.

after the initial burst if product is removed from the reaction. We therefore carried out a time course experiment in the presence of a thermophilic AP endonuclease. At 65 ◦ C, an oligonucleotide containing either a T–G mismatch or a central AP site should be stable. Once the strand containing the AP site is cleaved by the AP endonuclease, the 10-mer and the 9-mer annealed to the 19-mer should denature. Thus, AP endonuclease should remove any product that could inhibit Mig-Mth. The presence of 1.3 ␮M T. maritima

endonuclease IV changed the rate of production of product by Mig-Mth and allowed the reaction to go to completion (Fig. 2A, bottom; Fig. 2B, open squares). The same initial burst of product formation as seen for Mig-Mth alone was observed (Fig. 2C) with a kcat of 0.68 ± 0.13 min−1 (Table 2). After that, there was a second linear phase of product formation extending to approximately 15 min that was not seen with Mig-Mth alone (Fig. 2D, open squares). The slope of this line should reflect the rate of the uninhibited

Fig. 2. (A) Time course of Mig-Mth activity. Storage phosphor autoradiogram of a denaturing polyacrylamide gel containing samples in which enzyme was incubated with an oligonucleotide containing a T–G mismatch for 0–105 min. After incubation, the samples were treated with base to stop the reaction and cleave the phosphodiester backbone at AP sites. (Top) Mig-Mth alone, (bottom) Mig-Mth in the presence of a thermophilic AP endonuclease. (B) Extent of Mig-Mth activity with a T–G mismatch-containing oligonucleotide in the absence (closed circle) or presence (open square) of an AP endonuclease. The data from (A) and is plotted to show the extent of product formation seen under multiple-turnover conditions. (C) Initial burst of Mig-Mth activity with a T–G mismatch-containing oligonucleotide in the absence (closed circle) or presence (open square) of an AP endonuclease. The data from (A) is plotted to show the extent of product formation after 5 min. (D) The second phase of the Mig-Mth reaction under multiple-turnover conditions. The reaction is linear in the presence of an AP endonuclease (open square) but not in its absence (closed circle). (E) The initial burst of Mig-Mth activity proceeds up to 5 ␮M product (black arrow), which is also the concentration of Mig-Mth used in the absence (closed circle) and presence of an AP endonuclease (open square). Data from (A) is plotted to show that slope of the initial burst (solid line) is steeper than the second burst of activity (dashed line).

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Table 2 Mig-Mth kinetic constants determined under steady-state conditions

Mig-Mth Mig-Mth + endonuclease IV

kcat (min−1 )

Vss (phase-2) (␮M/min)

k(cat + 3) (min−1 )

k3 (min−1 )

0.83 ± 0.10 0.68 ± 0.13

– 1.40 ± 0.10

– 0.28 ± 0.02

– 0.47

steady-state reaction for Mig-Mth, Vss (Table 2). Because this reaction was carried out at a substrate concentration above Kd , the steady-state rate constant should reflect contributions from kcat and k3 . Thus: kss = kcat + k3

(3)

was present at either 1.3 ␮M or 90 ␮M. Neither concentration of endonuclease IV significantly affected kcat-st (Table 1) showing that endonuclease IV does not stimulate catalysis by Mig-Mth. 3.4. Binding of enzyme to product

and kcat+3 =

Vss [Mig-Mth]

(4)

The rate of product dissociation under steady-state conditions was determined using the equation: 1 1 1 = − k3 kcat+3 kcat

(5)

The rate of product dissociation was determined to be 0.47 min−1 , indicating that the enzyme remains associated with product for approximately 2 min after catalysis. A straight line could not be drawn through data generated with Mig-Mth alone. Mig-Mth was present at 5 ␮M and the initial burst of activity of Mig-Mth in the presence or absence of endonuclease IV yielded 5 ␮M product showing that the enzyme turned over once (Fig. 4E, black arrow). The decreased rate after this suggests that product release is rate limiting. The fact that we saw a linear phase for the steady-state reaction (Fig. 4E, dashed line) with product removal suggests that the rate of product binding, k−3 , is significant. 3.3. Effect of endonuclease IV on catalysis Endonuclease IV allowed the overall formation of AP sites by Mig-Mth to proceed more rapidly. This effect is clearly due to product removal but could also be due to interactions with Mig-Mth to accelerate the reaction. We performed single-turnover experiments with Mig-Mth and endonuclease IV to determine if the presence of endonuclease IV affected the kcat-st . Mig-Mth was present at 47 ␮M while endonuclease IV

We have previously shown that Mig-Mth binds to product, which is a duplex oligonucleotide containing an AP site opposite a guanine [12] and we have shown the same phenomenon with our kinetic experiments. Enzymes that recognize mismatches in DNA may be expected to look at both bases in the mismatch because a recognizable, damaged base is not present and recognition requires information from both bases to confirm a mismatch. We have systematically varied the base opposite an AP site in oligonucleotides to determine if the orphan base plays a role in the binding specificity of Mig-Mth for product. The ratio of DNA in a protein-DNA complex to the total amount of DNA was determined for concentrations of Mig-Mth ranging from 1 to 32 ␮M using EMSA (Fig. 3A). The product affinity at each Mig-Mth concentration was determined for each of the four product oligonucleotides. The percentage of free DNA was plotted as a function of Mig-Mth concentration using the program KaleidaGraph. Theoretical isotherms were calculated for each of the four AP site containing oligonucleotides using the following equation:
 [Mig-Mth] Y =1− (6) [Mig-Mth] + Kd where Y is the percentage of free product and Kd is the enzyme concentration at which 50% of the product is bound. The data were fit to isotherms for non-cooperative, single-site ligand binding (Fig. 3B) and a comparison of Mig-Mth binding to AP sites across from the four canonical bases shows that Mig-Mth exhibits the strongest binding to an oligonucleotide with a G opposite an AP site (Table 3),

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Fig. 3. (A) Sample storage phosphor autoradiogram of a representative binding experiment with a duplex oligonucleotide containing a product AP site opposite a guanine. (B) A theoretical binding isotherm (solid line) for Mig-Mth with an AP–G product was determined using Eq. (6) where the Kd is 1.6 ␮M and the data was fit to the data points derived from three experiments (open square).

Table 3 Binding constants for Mig-Mth to different product oligonucleotides Product

Kd (nM)

AP–guanine AP–thymine AP–cytosine AP–adenine

1600 2250 2750 6000

± ± ± ±

100 150 50 400

the physiological product of enzymatic activity. The binding of Mig-Mth to oligonucleotides containing AP sites shows the preference G > T > C > A for the orphan base.

Kd relative to AP–G – 1.41 1.72 3.75

3.5. Binding of enzyme to substrate We previously had measured binding of Mig-Mth to substrate using a catalytically inactive form of

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the enzyme, the mutant Y126K [12]. In order to use wild type enzyme for binding studies, we needed a non-cleavable substrate. Diflourotoluene (F) nucleoside is a non-hydrogen-bonding nucleoside shape analog that is isosteric with thymine. It behaves like thymine in vitro DNA replication systems and the F–A base pair is structurally very similar to a T–A base pair [28,29]. Difluorotoluene nucleoside contains a C1–C1 bond in place of the N1–C1 glycosidic bond of thymidine (Fig. 4A) and should be resistant to hydrolysis by DNA glycosylases. Mig-Mth was

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unable to catalyze the removal of this base from an F–G base pair in an oligonucleotide at concentrations well above those needed to efficiently catalyze N-glycosidic bond cleavage of thymines from a T–G mismatch (Fig. 4B). The binding affinity of Mig-Mth for an F–G containing 19-mer was determined using EMSA (Fig. 4C). In addition, the base opposite from F was systematically varied to include all the naturally occurring bases. The binding of Mig-Mth to oligonucleotides containing F shows the preference G > A > T > C for the opposite base (Table 4).

Fig. 4. (A) Structures of difluorotoluene and thymine nucleosides. (B) Storage phosphor autoradiogram showing the enzymatic activity of Mig-Mth on duplex oligonucleotides at 100 nM containing F–G and T–G mismatches. (C) Sample storage phosphor autoradiogram of a representative binding experiment with a duplex oligonucleotide containing difluorotoluene opposite a guanine.

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Table 4 Binding constants for Mig-Mth to different duplexes containing difluorotoluene Target

Kd (nM)

F–guanine F–adenine F–thymine F–cytosine

2550 5250 5550 8050

± ± ± ±

50 150 150 150

4. Discussion Several laboratories have noted a very low rate of catalytic activity of the pure glycosylases Mig-Mth [12], MutY [30–32] and MBD4 (MED1) [8]. In many reports it appears that MutY does not turn over at all. This type of behavior could represent enzyme inhibition by several different mechanisms including failure to release the hydrolyzed base from the active site pocket, covalent modification of the enzyme, or high affinity of the enzyme for DNA containing AP sites. We suspected that the low activity of Mig-Mth was due to its high affinity for product, and we utilized a kinetic analysis of the Mig-Mth reaction to examine this possibility in more detail. We examined the time-course of Mig-Mth-catalyzed base release to determine the maximum reaction velocity under single- and multiple-turnover conditions. The reaction rate under steady-state conditions was difficult to determine because the rate of product formation was not linear. We had previously determined that the binding constant for Mig-Mth to product was similar to that for substrate, and this can explain the lack of linearity in our assays. We examined the effect of product removal on the activity of Mig-Mth by including the AP endonuclease, endonuclease IV, in the enzyme reactions. A significant increase in glycosylase activity upon removal of the reaction product by endonuclease IV provides evidence that oligonucleotides containing AP sites act as inhibitors of Mig-Mth. The engineering of Mig-Mth to produce a mutant enzyme, Y126K, converted the glycosylase to an AP lyase [12]. The ability of this mutant enzyme to use an AP site as a substrate after the change of a single-catalytic residue also provides support for the idea that Mig-Mth binds to product in a similar fashion as to substrate. Single-turnover kinetic experiments were performed to determine the rate of catalysis by Mig-Mth. The rate constants from these experiments agree with

the rate constants derived from the initial burst observed under steady-state conditions. In both cases, the experiments were done with substrate concentrations in excess of Kd so that substrate binding should not be rate limiting. These experiments indicate that the catalytic step occurs relatively quickly. Knowing the catalytic rate allowed us to determine the off rate, koff , under steady-state conditions in the presence of endonuclease IV. The decrease in reaction velocity after the first catalytic event suggested that association with product plays a role in modulating the glycosylase activity of Mig-Mth. The fact that significant product inhibition is seen when substrate concentrations are higher than product concentrations suggests that Mig-Mth associates with AP sites in the reaction mixture to limit glycosylase activity and suggest that AP sites will be preferentially bound when compared to substrate. MutY also binds tightly to product AP sites. When the enzyme processes it natural substrate, which is an adenine opposite an 8-oxoguanine, it remains so tightly bound to product that no subsequent catalytic events are seen. Under these conditions, a steady-state cannot be established and conventional enzyme kinetics are not possible. When the enzyme processes an alternative substrate, adenine opposite a guanine, it is capable of carrying out multiple-catalytic events. In this case, the enzyme does not bind as tightly to product. Porello et al. [32] report that under multiple-turnover conditions, MutY exhibits an initial burst and then achieves a steady-state. Their data suggest that MutY is not as severely inhibited by this product (AP–G), when compared to its natural product (AP–8-oxoguanine). We compared the binding of Mig-Mth to oligonucleotides containing either an AP site opposite a guanine or a non-cleavable substrate analog, difluorotolune, opposite a guanine. The enzyme has a higher affinity for product than for the non-cleavable substrate analogue. We observed similar results when we used a catalytically inactive form of the enzyme with a normal T–G mismatch [12]. Both uracil glycosylase [33] and G:T/U mismatch-specific DNA glycosylase [34] have also been reported to bind to oligonucleotides containing AP sites more tightly than to their respective substrates. The association of Mig-Mth with product for a significant period of time after catalysis raises questions

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about the physiological importance of this association. This association could act as a means to prevent mutation. It has been shown that increased glycosylase activity without an increase AP endonuclease activity raises the spontaneous mutation rate [22]. Mig-Mth’s propensity to bind to product after catalysis and to rebind after the initial product dissociation event prevents the generation of new AP sites and effectively reduces the glycosylase activity. This suggests that Mig-Mth will not create a new AP site until the old one has been processed by the BER pathway. Product binding could also constitute a way in which the cell protects AP sites from detrimental interactions with enzymes not involved in base excision repair. Topoisomerases have been shown to form covalent complexes with AP sites and to cause double strand breaks that lead to cell death [35,36]. The removal of topoisomerase-DNA cross-links has been recently shown to be catalyzed by a Tyr-DNA phoshodiesterase found in yeast, representing another important cellular DNA repair activity [37]. Steric protection of AP sites by Mig-Mth could exclude cellular enzymes that interact with these lesions in a non-productive manner, and could provide a damage avoidance function for DNA glycosylases. Product binding could also be used as a signal for binding of the next enzyme in the BER pathway [38]. Human uracil glycosylase and human thymine-DNA mismatch glycosylase activities have been shown to increase in the presence of HAP1 (human AP endonuclease) [33,39] suggesting product binding can be used to coordinate hand-offs between members of the base excision repair pathway. Dissociation of Mig-Mth from an AP–G product was analyzed in the context of the initial steps of a high-temperature base excision repair pathway. The only characterized thermophilic 5 AP endonuclease is Tma endonuclease IV [27] and we chose to analyze the Mig-Mth reaction in the presence of this enzyme. Our burst kinetics data suggest that endonuclease IV had no effect on product dissociation, similar to results observed using human TDG and endonuclease IV. In vivo the rate of product dissociation should be more rapid to allow for the efficient repair of these AP sites formed by glycosylase action. The increase in the rate of dissociation might require species specific protein contacts not found using Tma endonuclease IV and Mig-Mth, or contacts

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from other components of the base excision repair pathway; a dRpase, DNA polymerase and/or a DNA ligase in addition to an AP endonuclease. The idea that protein–protein interactions induce dissociation leads to the suggestion that a DNA repairosome may exist for BER [40–43]. Protein complexes provide a method to efficiently process a macromolecule through a cellular pathway, while excluding it from other pathways. It is reasonable to believe that a repairosome would be employed in the base excision repair pathway to correct pre-mutagenic DNA lesions, and exclude certain enzymes that could interact non-productively with AP sites. Steady-state kinetic experiments that monitor the second burst of the Mig-Mth reaction time course could than be used to identify proteins that interact with this glycosylase to increase its rate of product dissociation. The interaction of Mig-Mth with product raises the question of whether the orphan base opposite the AP site affects binding. Structural analysis of 5-methylcytosine DNA methlytransferase has shown that nucleotide flipping is a method by which these enzymes modify target bases. The flipping reaction has been theorized to push the target base out of the plane of the double helix by intercalation of protein subunits that interact with the orphan base in some fashion [44]. This mechanism has also been proposed for substrate recognition by DNA glycosylases [1,45–50]. Amino acid residues that interact with the orphan base could also help determine the specificity of substrate recognition. Using gel retardation experiments we have examined the effect of the orphan base on product binding. We altered the base opposite the AP site so that all the four naturally occurring DNA bases could be compared for effects on binding to Mig-Mth. The experiment clearly shows that Mig-Mth has the highest affinity for an orphan guanine, with order of affinity for the orphan base being G > T > C > A, suggesting that specific contacts between Mig-Mth and the orphaned guanine exist. The fact that the orphan guanine affects binding to product suggests that it may also affect Mig-Mth’s interaction with substrate. This hypothesis was tested using oligonucleotides containing the thymine analogue, F. F has the same shape as thymine but it does not have the ability to hydrogen bond at the 2, 3 or 4 positions. An F–adenine base pair results in a reduced thermal stability when compared to normal

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base pairs but it offers a similar shape and geometry as a T–A. F has been shown to normally base pair with adenine during DNA replication, adding support to the idea that shape complimentarity plays an important role in replication [29]. An F–G duplex was tested as a substrate for Mig-Mth and the C –C bond which replaces the C –N glycosidic bond was not cleaved by Mig-Mth. Oligonucleotides containing F provide a non-cleavable substrate for binding studies with Mig-Mth. We examined the effect of the base opposite F on Mig-Mth binding. We determined that Mig-Mth has the following hierarchy of binding affinity: G > C ∼ = T > A. The fact that the opposite strand does affect the binding affinity for Mig-Mth to a diflourotoluene containing duplex, suggests that specific contacts are made with the G opposite the target base. Structural work performed on the mismatch specific uracil glycosylase from E. coli (MUG) with product DNA has shown direct contacts with this orphan base [34]. We would predict the same for a Mig-Mth–DNA complex. The base opposite the target would then be a specificity determinant for Mig-Mth. Clearly other factors will be involved in substrate recognition, and these are under investigation. In conclusion, the kinetic analysis of Mig-Mth alone, and as part of a base excision repair pathway, supports the idea that product AP sites are bound tightly by Mig-Mth and regulate its activity. Product binding and endonuclease mediated stimulation have been reported in studies for human OGG1, human TDG and E. coli MutY [51–54] and together with our data, we believe this enzyme behavior can viewed as a built in mechanism to prevent aberrant activity. Further, interactions between Mig-Mth and the orphan base across from AP sites and F containing oligonucleotides have been shown to affect enzyme affinity for product and “substrate”, and suggest that the orphan base can be used to modulate the activity of this mismatch glycosylase.

Acknowledgements We would like to thank Dr. Charles Scholes and Alex Shektman for their help. This work was supported by NIH grant GM46312.

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