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DNA Repair 10 (2011) 545–553

Contents lists available at ScienceDirect

DNA Repair

journa l homepage: www.e lsev ier .com/ locate /dnarepai r

ependence of substrate binding and catalysis on pH, ionic strength, andemperature for thymine DNA glycosylase: Insights into recognition androcessing of G·T mispairs

tanu Maiti, Alexander C. Drohat ∗

epartment of Biochemistry and Molecular Biology and Greenebaum Cancer Center, School of Medicine, University of Maryland, 108 N Greene St., Baltimore, MD 21201, USA

r t i c l e i n f o

rticle history:eceived 20 December 2010eceived in revised form 4 March 2011ccepted 8 March 2011vailable online 6 April 2011

eywords:ase excision repair-Methylcytosine deamination·T mispairpG sitesluorescence

a b s t r a c t

Repair of G·T mismatches arising from deamination of 5-methylcytosine (m5C) involves excision ofthymine and restoration of a G·C pair via base excision repair (BER). Thymine DNA glycosylase (TDG)is one of two mammalian enzymes that can specifically remove thymine from G·T mispairs. While TDGcan excise other bases, it maintains stringent specificity for a CpG context, suggesting deaminated m5Cis an important biological substrate. Recent studies reveal TDG is essential for embryogenesis; it helps tomaintain an active chromatin complex and initiates BER to counter aberrant de novo CpG methylation,which may involve excision of actively deaminated m5C. The relatively weak G·T activity of TDG has beenimplicated in the hypermutability of CpG sites, which largely involves C → T transitions arising from m5Cdeamination. Thus, it is important to understand how TDG recognizes and process substrates, particu-larly G·T mispairs. Here, we extend our detailed studies of TDG by examining the dependence of substratebinding and catalysis on pH, ionic strength, and temperature. Catalytic activity is relatively constant forpH 5.5–9, but falls sharply for pH > 9 due to severely weakened substrate binding, and, potentially, ion-ization of the target base. Substrate binding and catalysis diminish sharply with increasing ionic strength,particularly for G·T substrates, due partly to effects on nucleotide flipping. TDG aggregates rapidly and

irreversibly at 37 ◦C, but can be stabilized by specific and nonspecific DNA. The temperature dependence ofcatalysis reveals large and unexpected differences for G·U and G·T substrates, where G·T activity exhibitsmuch steeper temperature dependence. The results suggest that reversible nucleotide flipping is muchmore rapid for G·T substrates, consistent with our previous findings that steric effects limit the active-

whictalysi

site lifetime of thymine,important insight into ca

. Introduction

While the repair of G·T mismatches generated by replicationrrors is directed at the new strand, repair of those caused by deam-nation of 5-methylcytosine (m5C) must involve the removal of Tnd restoration of a G·C base pair. Two mammalian enzymes exhibit

pecificity for excising T (but not G) from G·T mispairs, thymineNA glycosylase (TDG) [1,2], the focus of this work, and methylinding domain IV (MBD4) [3,4]. Downstream base excision repairroteins complete the process of restoring a G·C base pair. TDG

Abbreviations: AP, apurinic/apyrimidinic; BER, base excision repair; CD, circularichroism; dU, 2′-deoxyuridine; dT, 2′-deoxythymidine; HPLC, high pressure liquidhromatography; TDG, thymine DNA glycosylase; m5C, 5-methylcytosine; MBD4,ethyl binding domain IV; MUG, mismatch-specific uracil DNA glycosylase; UNG,

racil DNA glycosylase.∗ Corresponding author. Tel.: +1 410 706 8118.

E-mail address: adrohat@som.umaryland.edu (A.C. Drohat).

568-7864/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.dnarep.2011.03.004

h may account for the relatively weak G·T activity. Our findings provides by TDG, particularly for mutagenic G·T mispairs.

© 2011 Elsevier B.V. All rights reserved.

can also remove a broad range of other bases from DNA in vitro(uracil, 5-halouracils, among others), often with greater efficiencythan it exhibits for G·T mispairs [5,6]. However, without excep-tion, mammalian TDG exhibits stringent specificity for removingbases that are paired with guanine rather than adenine and that arelocated in a CpG dinucleotide context [7–10]. Of the many basesthat can be efficiently removed by TDG, only deaminated m5C islikely to arise selectively in a CpG context. These findings sug-gest G·T mispairs arising by m5C deamination are an importantbiological substrate for TDG [9]. This conclusion is supported bystudies of MBD4 deficient mice, which reveal an increase in C → Ttransitions at CpG sites but also indicate that factors other thanMBD4 contribute to the repair of deaminated m5C [11,12]. As men-tioned above, TDG is the only other mammalian enzyme known to

have specificity for initiating repair of G·T mispairs created by m5Cdeamination.

Many studies indicate a role for TDG in regulating gene expres-sion, and this appears to involve both its catalytic activity andinteractions with proteins involved in transcriptional regulation.

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46 A. Maiti, A.C. Drohat / D

ytosine methylation (m5C) is an epigenetic signal for gene silenc-ng, and maintaining the appropriate methylation status of CpGinucleotides and chromatin structure is essential for proper reg-lation of gene expression and for embryonic development [13].hile it is established that cytosine 5-methyltransferases con-

ert C to m5C, the mechanism for reversing this modification hasemained unclear. Previous studies suggest a role for TDG and BERn the active demethylation of CpG sites, which may involve exci-ion of deaminated m5C by TDG [14–18]. A recent study revealsDG is essential for embryonic development and indicates it isequired for maintaining epigenetic stability of genes expressedn development, by contributing to the assembly of chromatin

odifying complexes and by initiating BER to counter aberrante novo CpG methylation [19]. The requirement for TDG cat-lytic activity seems likely to involve excision of deaminated m5C,ecause TDG has exceedingly weak activity for removing m5C [19].emethylation could potentially involve conversion of m5C to 5-ydroxymethylcytosine (hmC) by TET proteins [20]. Our previousork suggests TDG would also have weak activity for excising hmC,

hough it has substantial activity for excising the deamination prod-ct of hmC, 5-hydroxymethyluracil, particularly from a CpG context6].

Together, these findings indicate the importance of obtainingdetailed understanding of how TDG recognizes and processes

ubstrates, particularly G·T mispairs. Deamination of m5C is con-idered a major source of C → T transitions at CpG sites, which aremong the most frequent point mutations in cancer and geneticisease [21–24]. While the findings above suggest G·T mispairsrising from m5C deamination are an important biological sub-trate of TDG, many studies have shown that binding and catalysiss substantially weaker for G·T mispairs relative to other sub-trates identified by in vitro studies [8,10,25,26]. It is importanto understand why this is the case, to further our knowledgef the TDG catalytic mechanism, and because it may be rele-ant to the high mutational frequency observed at CpG sites27].

Unlike the vast majority of DNA glycoyslases, TDG and MBD4ace the formidable challenge of removing a normal base from a

ismatched base pair. The relatively weak G·T activity exhibited byhese enzymes might reflect a compromise between the need forfficient processing of deaminated m5C and for avoiding aberrantemoval of T from A·T pairs [11,27]. Consistent with this hypothe-is, our previous findings suggest the TDG active site offers limitedccess to thymine and other bases with a bulky C-5 substituent,ue likely to steric hindrance [6,10,26,28]. Indeed, our findings

ndicate that the much lower catalytic activity observed for G·Telative to G·U substrates can be largely explained by steric hin-rance involving the methyl group of thymine, although stabilityf the scissile N-glycosylic bond is likely a contributing factor, ashe non-enzymatic hydrolysis is ∼3-fold faster for dU relative toT (at 22 ◦C) [6,29]. Likewise, steric effects seem likely to account

argely for the 30-fold weaker binding affinity of TDG for G·T rel-tive to G·U substrates [28]. Moreover, the much lower catalyticctivity for the excision of 5-BrU versus 5-FU can be attributedargely to steric effects involving the C-5 substituent, because the-glycosylic bond is of nearly equivalent stability for BrdU and FdU

6,10].Here, we extend our detailed studies of how TDG recognizes

nd processes its substrates, particularly G·T and G·U mispairs,nd further explore the molecular basis of its relatively weak G·Tctivity. We rigorously examined the effects of varying pH, ionictrength, and temperature on binding and catalysis for G·T and G·U

ubstrates, using single turnover kinetics and equilibrium bindingxperiments (monitored by fluorescence anisotropy). Such studiesave not previously been reported for any member of the largeDG–MUG family of enzymes. The pH dependence of substrate

air 10 (2011) 545–553

binding and catalysis can provide important insight into enzymemechanism [30], as shown for uracil DNA glycosylase (UNG) andalkyladenine DNA glycosylase (AAG) [31,32]. We find dramatic andunexpected differences in the temperature dependence of catal-ysis for G·T and G·U substrates, which are consistent with thehypothesis that G·T activity is limited by steric effects involving themethyl group of thymine. While many previous studies of TDG havebeen performed at physiological temperature, the stability of TDGat 37 ◦C had not been established. Accordingly, we examined thethermal stability of TDG using CD and fluorescence spectroscopy,and in the presence and absence of specific and nonspecific DNA.The findings have important implications for obtaining experimen-tal conditions that maintain enzyme activity, and conditions thatprovide maximal activity, including considerations of temperature,ionic strength, and the presence of DNA. Together, our findings pro-vide important new insight into the catalytic mechanism of TDG,including recognition and processing of G·T mispairs arising fromm5C deamination.

2. Materials and methods

2.1. DNA synthesis and purification

The 16 bp DNA construct, used for most experiments,contained 5′-CTCAxGTACAGAGCTG, where x is the targetnucleotide (x = T, TF, U, UF), and 5′-CAGCTCTGTACGTGAG, suchthat the target nucleotide is paired with G and located inCpG context (Supplementary Fig. 1). Similarly, the 28 bp DNAcontained 5′-GTGTCACCACTGCTCAxGTACAGAGCTG and 5′-CAGCTCTGTACGTGAGCAGTGGTGACAC. The 32 bp G·T substratehas the same construct as the 28 bp DNAs, with GC nucleotidesadded at each terminus. The 28 bp nonspecific DNA (NS28) isnearly identical but does not contain a mismatch or a CpG site.Oligonucleotides were synthesized and purified as described [26],with purity verified by ion-exchange HPLC [6]. Duplex DNA washybridized by rapid heating to 80 ◦C followed by slow cooling toroom temperature. DNA used for fluorescence anisotropy waslabeled with sulforhodamine (Texas Red, TR) in the non-targetstrand (5′ amino C6 modifier) [28], which was synthesized andpurified (RP-HPLC) by Midland Certified Reagent Co. (Midland,TX). Oligonucleotides containing the dT analog 2′-deoxy-2′-flouroarabinothymidine (TF, Supplementary Fig. 1) and the dUanalog 2′-deoxy-2′-flouroarabinouridine (UF) were obtained asdescribed [26]. Control experiments demonstrate DNA containingTF or UF is completely resistant to cleavage by TDG [26], consistentwith previous findings [33,34]. These analogs differ from thenatural dT and dU nucleotides only by replacement of 2′-H withfluorine (2′-fluoroarabino), which renders the N-glycosylic bondof these and other nucleotides highly resistant to enzymaticcleavage [26,31,33,34]. Previous studies show the 2′-fluoroarabinosubstitution, in dT and other deoxynucleotides, promotes anO4′-endo sugar pucker, which is fully compatible with B-DNAgeometry [35–37]. Although the O4′-endo conformation of TF

could potentially alter TDG binding (relative to binding for nat-ural dT), our previous studies indicate any such effect is small[26].

2.2. Enzyme purification

Human TDG was expressed and purified as previously described

flash frozen, and stored at −80 ◦C. TDG (56–308) was purified essen-tially as previously described for TDG (111–308) [38], quantifiedby absorbance (ε280 = 17.4 mM−1 cm−1), flash frozen, and stored at−80 ◦C.

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.3. Kinetics experiments

Kinetics experiments were collected under single turnover con-itions ([TDG] � [substrate]) in order to eliminate effects of productelease or product inhibition on the observed rate constants (kobs).his is important because TDG exhibits very slow product releasend strong product inhibition, which dominates kcat values deter-ined under steady-state (multiple turnover) conditions [8,25,39].

xperiments were collected in HEMN.1 buffer (0.01 M HEPES,.2 mM EDTA, 2.5 mM MgCl2, and 0.1 M NaCl) at 22 ◦C, unless oth-rwise noted. Experiments were initiated by adding concentratedDG to buffered substrate, followed by rapid mixing. At variousime points, aliquots were removed, quenched with 50% (v:v) 0.3 MaOH and 0.03 M EDTA, and heated at 85 ◦C for 15 min to cleave theNA backbone at enzyme-produced abasic sites. The fraction prod-ct was determined by HPLC [6,10], and data were fitted to Eq. (1)sing non-linear regression with Grafit 5 [40]:

raction product = A(1 − e−kt) (1)

here A is the amplitude, k is the observed rate constant, and t ishe reaction time (min).

.4. Equilibrium binding monitored by fluorescence anisotropy

We used fluorescence anisotropy to determine equilibrium dis-ociation constants for TDG binding to G·TF and G·UF substratenalogs (10 nM and 0.5 nM, respectively) as described [28], mon-toring the fluorescence of sulforhodamine (Texas Red, or TR) athe 5′-end of the non-target DNA strand, using a QuantaMaster0 spectrofluorometer (PTI, Birmingham, NJ). Equilibrium dissoci-tion constants for TDG binding to the G·TF substrate analog wereetermined by fitting anisotropy data from at least two indepen-ent binding experiments using DynaFit 4 [41,42] as previouslyescribed [28] and detailed below. Our previous studies show TDGinds DNA containing a G·T substrate analog initially with 1:1 sto-

chiometry and a second TDG subunit can bind for high and excessDG concentrations, giving a 2:1 complex [28]. The same behaviors observed for TDG binding to DNA containing other specific sitesG·U mispair, G·AP product, undamaged CpG site) [28]. A benefitf using DynaFit for fitting data to complex binding models is thattting does not require an analytical equation, and experimentsre not limited by restraints imposed by assumptions associatedith an analytical equation (i.e., the concentration of labeled DNA

s far below Kd). Thus, binding experiments can be collected usingconcentration of labeled DNA that approximates the Kd, even

or complex 2:1 binding models. A representative example of thecripts used for data fitting, including the 1:1 and 2:1 bindingodels, is provided in Supplementary Data. The fitted parame-

ers include the dissociation constants (Kd1, Kd2) and anisotropyalues for free DNA (rD) and enzyme–DNA complexes with stoi-hiometry of 1:1 (rED) and 2:1 (rEED). We fitted the data using axed value of rED (0.20), because if rED is not fixed, Kd2 and rED areoorly constrained and unreasonably high. The choice of rED = 0.20

s based on the rED value obtained from fitting anisotropy dataor TDG binding to similar DNA containing a G·U or a G·AP sitefor which all parameters are well constrained) [28]. In all cases, auperior fit was obtained for the 2:1 rather than 1:1 model, basedn visual inspection and/or the probability value of the Fisher’s-statistic (obtained from fitting), where p < 0.05 is consideredignificant.

.5. Fluorescence spectroscopy to monitor conformationalhanges in TDG

Fluorescence emission spectra of TDG were collected in vari-us buffers (below), to monitor the effects of temperature, pH, and

air 10 (2011) 545–553 547

ionic strength on the structure of TDG. An excitation wavelength of295 nm was used to excite Trp and minimize excitation of Tyr [43],because the 15 Tyr residues of TDG contribute strongly to fluores-cence for excitation at 280 nm. Emission spectra for TDG (1 �M)were typically collected over the range of 310 nm to 410 nm, at1 nm intervals with integration time of 1 s. Spectra collected to mea-sure effects of pH or ionic strength are the average of two scans. Toallow rapid data acquisition for experiments at 37 ◦C, spectra wererecorded from 315 nm to 365 nm with a 0.2 s integration time.

2.6. Circular dichroism (CD) spectroscopy

Thermal melting experiments for TDG (2.5 �M) were conductedwith a Jasco J-715 spectropolarimeter (Easton, MD) with a Peltierfor temperature control and a 1 cm path length cuvette, monitor-ing ellipticity at 222 nm (CD222). Data were collected from 10 ◦C to60 ◦C, with a temperature gradient of 1 ◦C per min. The buffer was0.01 M Tris–HCl pH 7.5, 0.1 M NaCl, and 0.5 mM DTT. In additionto CD222, the spectropolarimeter simultaneously records the highvoltage applied to dynodes of the photomultiplier (PM voltage).The PM voltage increases with a reduction in light intensity, due toabsorption and/or scattering. Because ε222 exhibits small temper-ature dependence, no significant change in absorption is expected,and changes in PM voltage are attributed to light scattering [44,45].

2.7. Buffers for studying the effects of pH and ionic strength onTDG structure and activity

The effect of pH on G·T substrate binding, catalytic activity,and TDG structure (Trp fluorescence) was determined using a0.01 M concentration of an appropriate buffer (see below) supple-mented with 0.1 M NaCl, 2.5 mM MgCl2, and 0.2 mM EDTA. Thebuffers used for various pH values were [31]: sodium phosphate,pH 2; sodium acetate, pH 4–5.5; NaMES, pH 5.5–6.8; NaHEPES, pH6.8–8.0; Tris–HCl, pH 8.0–9.0; NaCHES, pH 9.0–10.0; NaCAPS, pH10.0–11.0; sodium phosphate, pH 12 (without MgCl2 due to for-mation of insoluble magnesium hydroxide). Buffers for studyingthe effect of increasing ionic strength on binding and catalysis con-tained 0.01 M HEPES (pH 7.5), 2.5 mM MgCl2, 0.2 mM EDTA, andsufficient NaCl to give the desired ionic strength.

3. Results

3.1. Experimental approach

Previous kinetics studies of TDG and MUG enzymes show thatproduct release is very slow and product inhibition is potent, suchthat kcat values obtained from steady-state kinetics are dominatedby steps that occur after the chemical step (kchem) [25,39,46].Accordingly, we used single turnover kinetics experiments, whichyield rate constants (kobs) that report on nucleotide flipping (Kflip)and kchem, but are not influenced by steps after chemistry [6,25].Nucleotide flipping is a conformational change used by glycosy-lases whereby the target nucleotide flips out of the DNA and intothe enzyme active site to give the reactive (Michaelis) complex.We used 16 bp G·U and G·T substrates, with the mispair in a CpGsequence context, consistent with TDG specificity (SupplementaryFig. 1). The 16 bp substrates used here satisfy all TDG–DNA inter-actions observed in a crystal structure of TDG (catalytic domain)bound to DNA, and they provide full catalytic activity under sat-urating enzyme conditions, consistent with our previous results

demonstrating that 2:1 binding does not contribute to catalyticactivity for G·T or G·U substrates [28,38]. However, because thebinding affinity is somewhat tighter for 28 bp relative to 16 bp G·Tsubstrates [28], due likely to nonspecific interactions involving thedisordered N-terminal region of TDG [47], we used a longer G·T sub-

548 A. Maiti, A.C. Drohat / DNA Repair 10 (2011) 545–553

Fig. 1. pH dependence of catalysis and substrate binding. (A) Single turnover kinetics experiments were collected (at 22 ◦C) to determine the rate of base excision (kobs) forTDG (2.5 �M) acting upon 16 bp G·U (©) or G·T () substrates (0.25 �M) as a function of pH. The rate was also determined at pH 9 (blue �) using 8-fold higher concentrationsof TDG (20 �M) and 16 bp G·T substrate (2 �M). Data are also shown for a 32 bp G·T substrate (0.25 �M), collected with 2.5 �M TDG (red ♦). Buffers are given in Section 2 (ionicstrength was 0.11 M). kobs values represent the mean of at least three independent experiments. (B) Equilibrium binding of TDG to a G·TF DNA substrate analog (10 nM) at 22 ◦C,and pH 7.5 (©), pH 8.5 (�) or pH 9.5 (), monitored by fluorescence anisotropy using sulforhodamine labeled G·TF DNA. Data from two independent experiments were fittedt follop d2 = 22w ysiolot

sr

sstFflrcpobo((stsb

3

usci(g2to

cpiiGoaoc

o a two-site binding model, as described previously and in Section 2 [28], giving theH 8.5 (�), Kd1 = 0.42 ± 0.05 �M, Kd2 = 3.1 ± 0.3 �M; pH 9.5 (), Kd1 = 4.4 ± 0.5 �M, Kith weak affinity at high and excess concentrations of TDG, and is probably not ph

he reader is referred to the web version of the article.)

trate for some experiments, which provided essentially the sameesult as the 16 bp G·T substrate.

To study the effects of pH and ionic strength on TDG binding toubstrates in the absence of base cleavage, we used G·TF and G·UF

ubstrate analogs, where TF and UF are close mimics of dT and dUhat bind specifically but are not cleaved by TDG (Supplementaryig. 1) [26,33]. The equilibrium binding experiments, monitored byuorescence anisotropy, provide a dissociation constant (Kd1) thateports on at least two steps, formation of the initial TDG–DNAomplex (KES) and nucleotide flipping (Kflip) [28]. As we have shownreviously, TDG can form a 2:1 complex with DNA under conditionsf high and excess concentrations of TDG, where one TDG subunitinds the specific site (i.e., G·TF) with high affinity (Kd1) and a sec-nd TDG subunit binds nonspecifically with much weaker affinityKd2) [28,38]. Our studies also showed that under limiting enzymemultiple turnover) conditions, TDG binds and processes G·T sub-trates using 1:1 stoichiometry. Accordingly, our interest here ishe effect of pH and ionic strength on the affinity of TDG for thepecific site (Kd1) rather than the nonspecific site (Kd2), althoughoth are reported.

.2. pH dependence of catalysis

We determined the pH dependence of catalysis for TDG actingpon G·T and G·U substrates using single turnover experiments, ashown in Fig. 1A. TDG activity for the G·U substrate is relativelyonstant for pH 5.5-9.0, falls 90-fold for pH 9.0-10, and no activitys detected at pH 10.5. At pH 5.0, G·U activity is observed initiallyup to 20 s) but it dissipates before the reaction is complete, sug-esting an inactivating TDG conformational change occurs (within0 s) at pH 5.0, consistent with results shown below. This is not dueo an effect of the buffer (sodium acetate), because full activity isbserved in the same buffer at pH 5.5.

For the G·T substrate and a fixed (2.5 �M) concentration of TDG,atalytic activity is fairly constant for pH 6.5–7.5, falls 6-fold fromH 7.5–9, and activity is not detected at pH 9.5. For pH 5.5, activity

s observed initially (up to 2 min) but dissipates before the reactions complete, indicating a slow loss of TDG activity at pH 5.5. (The

·U reaction is complete in <1 min at pH 5.5). No G·T activity isbserved at pH 5.0, consistent with results for G·U. Similar resultsre obtained for a longer G·T substrate (Fig. 1A). Notably, the lossf G·T activity at pH 9 can be largely recovered by using a higheroncentration of TDG (Fig. 1A), consistent with findings below that

wing dissociation constants: pH 7.5 (©), Kd1 = 0.12 ± 0.02 �M, Kd2 = 0.98 ± 0.17 �M;± 5 �M. As seen here and discussed previously [28], the second TDG subunit bindsgically relevant. (For interpretation of the references to color in this figure legend,

substrate binding is severely weakened with increasing pH. Whenthe data collected at pH 9 with a higher TDG concentration areconsidered, the pH profiles for G·T and G·U activity are quite similarover the range of pH 6–9 (Fig. 1A).

3.3. pH dependence of substrate binding

To examine the role of substrate binding in the loss of G·T activ-ity for pH > 7.5, we performed equilibrium binding experiments,monitored by fluorescence anisotropy with a labeled G·TF substrateanalog, as shown in Fig. 1B. At pH 7.5, TDG binds the G·TF analogwith a dissociation constant of Kd1 = 0.12 ± 0.02 �M. The bindingaffinity is nearly 4-fold weaker at pH 8.5, Kd1 = 0.42 ± 0.05 �M,and 36-fold weaker at pH 9.5, Kd1 = 4.4 ± 0.5 �M. A very simi-lar result is observed for a longer G·T substrate (SupplementaryFig. 2A). We also examined the effect of increasing pH on G·Usubstrate binding. At pH 7.5, TDG binds a G·UF analog tightly,Kd1 = 0.008 ± 0.003 �M [28], but the affinity is 30-fold weaker atpH 9.0, Kd1 = 0.244 ± 0.046 �M (Supplementary Fig. 2B). While G·Usubstrate binding is much weaker at pH 9.0, the concentration ofTDG used in the kinetics experiments above (2.5 �M) is sufficient tosaturate the substrate such that activity is not diminished (Fig. 1A).In contrast, G·T substrate binding is sufficiently weakened to affectG·T catalytic activity for pH > 7.5–9, consistent with the finding thatG·T activity can be recovered at pH 9.0 by using a higher concen-trations of TDG (Fig. 1A).

3.4. Effects of varying pH on TDG structure

When catalytic activity decreases at high or low pH, it is impor-tant to determine whether the loss may be due, at least in part, tostructural perturbations of the enzyme. We used tryptophan fluo-rescence to monitor pH-induced conformational changes in TDG.The emission energy (�max) of Trp is highly sensitive to polarity;�max is about 355 nm for Trp in water, while �max can be as lowas 310 nm for Trp buried in the hydrophobic interior of a pro-tein [43,48]. Protein unfolding invariably leads to an increase in�max for a buried Trp, while the effect on quantum yield (inten-

sity, Imax) is less predictable [43]. TDG contains two Trp residues,Trp160, buried in the catalytic domain [38,49], and Trp383, in the C-terminal region, which is intrinsically disordered [50]. As indicatedin Supplementary Fig. 3, emission from Trp383 dominates the flu-orescence of TDG. Thus, to focus on pH-induced conformational

A. Maiti, A.C. Drohat / DNA Repair 10 (2011) 545–553 549

F p fluorp in theN ntaryi

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ig. 2. Effect of varying pH on TDG structure monitored by Trp fluorescence. (A) TrH indicated by color. TDG (56–308) contains a single Trp residue, Trp160, buriedote that in 6 M Gdn–HCl, �max = 350 nm for both TDG (56–308) and TDG (Suppleme

s referred to the web version of the article.)

hanges in the catalytic domain, we used TDG (56–308), whichontains only Trp160. Importantly, TDG (56–308) exhibits essen-ially the same substrate binding and catalytic activity as full-lengthDG [47,50–52]. As shown in Fig. 2, fluorescence spectra for TDG56–308) at pH 7.5 reveal Trp160 exhibits �max = 315 nm, indicat-ng a rather hydrophobic environment. �max is relatively constantor pH 6–9, suggesting no substantial conformational change, con-istent with the flat pH profile for pH 6–9. The small increase inmax and changes in Imax for pH 5.5, 9.5, and 10 suggest a modestonformational change (Supplementary Fig. 3D), consistent withiminished G·U activity and absence of G·T activity for pH > 9, and

oss of activity within minutes at pH 5.5. Large increases in �max

and changes in Imax) for pH ≤ 5 and pH ≥ 10.5 indicate unfoldingf TDG, consistent with the absence of catalytic activity at these pHxtremes.

.5. Dependence of substrate binding and catalysis on ionictrength

We also examined the effect of increasing ionic strength on TDGctivity. As shown in Fig. 3A, activity for G·U substrates is relatively

onstant for ionic strength of 0.01–0.2 M, falls dramatically as ionictrength increases to 0.4 M, and no activity is detected at 0.6 M ionictrength. The effects of increasing ionic strength are even larger for·T activity. Relative to the activity at 0.06 M ionic strength, activity

s modestly lower at 0.11 M, 4-fold lower at 0.16 M, 10-fold lower

ig. 3. Catalytic activity and substrate binding are sharply diminished by increasing ionetermine the base excision (kobs) for TDG (5 �M) and G·U (©) or G·T () substrate (0.5

onic strength (blue �) using 4-fold higher concentrations of TDG (20 �M) and G·T subst�M TDG (red ♦). (B) Equilibrium binding of TDG to G·TF DNA substrate analog (10 nM♦), monitored by fluorescence anisotropy (sulforhodamine labeled G·TF DNA) at 22 ◦C.

odel with two binding sites for TDG (Section 2), giving the following parameters: 0.06 Md2 = 0.98 ± 0.2 �M; 0.16 M (), Kd1 = 1.1 ± 0.1 �M, Kd2 = 10.5 ± 1.3 �M. (For interpretationersion of the article.)

escence spectra of TDG (56–308) (1 �M) collected at 22 ◦C in varying buffers, withcatalytic domain. (B) �max as a function of pH for TDG (56–308) (©) and TDG ().Fig. 3). (For interpretation of the references to color in this figure legend, the reader

at 0.2 M, 120-fold lower at 0.3 M, and activity is not detected at0.4 M. The results are essentially the same for a 32 bp G·T substrate(Fig. 3A). The loss in G·T activity at 0.16 M ionic strength is not recov-ered by increasing the concentration of TDG by 4-fold (Fig. 3A),indicating that increasing ionic strength impairs nucleotide flip-ping and/or the chemical step of the TDG reaction, in addition toeffects on the initial association of TDG and substrate (as discussedbelow). Fluorescence studies indicate the diminished activity is notdue to substantial conformational changes induced by increasingionic strength (Supplementary Fig. 4), as expected. Notably, freeTDG aggregates at very low ionic strength, 0.01 M (SupplementaryFigs. 4 and 5), but observation that it retains full catalytic activityat this ionic strength indicates that binding to DNA substrate (orproduct) suppresses aggregation at low ionic strength.

To assess the extent to which the loss in catalytic activity withincreasing ionic strength is due to an effect on substrate bind-ing, we examined the dependence of substrate binding on ionicstrength, using fluorescence anisotropy. As shown in Fig. 3B, theaffinity of TDG for a G·TF substrate analog depends strongly on ionicstrength. TDG exhibits high affinity for the G·TF analog at 0.06 Mionic strength, Kd1 = 0.024 ± 0.003 �M, but the affinity is 5-fold

weaker at 0.11 M, Kd1 = 0.12 ± 0.02 �M, 46-fold weaker at 0.16 M(near physiological), Kd1 = 1.1 ± 0.1 �M, and exceedingly weak at0.3 M. A very similar result is observed for a longer G·T substrate(Supplementary Fig. 4A). Together, the binding and kinetics resultsindicate the effect of increasing ionic strength on catalytic activity

ic strength. (A) Single turnover kinetics experiments were collected (at 22 ◦C) to�M) in buffers of varying ionic strength. The rate was also determined at 0.16 M

rate (2 �M). Data are also shown for a 32 bp G·T substrate (0.5 �M) collected with) in buffers with ionic strength of 0.06 M (�), 0.11 M (©), 0.16 M (), and 0.31 MFor each buffer, data from two independent binding experiments were fitted to a(�), Kd1 = 0.024 ± 0.003 �M, Kd2 = 0.54 ± 0.09 �M; 0.11 M (©), Kd1 = 0.12 ± 0.02 �M,of the references to color in this figure legend, the reader is referred to the web

550 A. Maiti, A.C. Drohat / DNA Rep

Fig. 4. Temperature dependence of catalytic activity for TDG. Single turnover kinet-ics experiments were used to determine the rate of base excision for TDG (5 �M)as a function of temperature for G·U (©) and G·T () substrates (0.5 �M). We alsodetermined the rate using 4-fold higher concentrations of TDG (20 �M) and G·T sub-s(c

i(

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(Fig. 1B, Supplementary Fig. 2). Contributing factors could also

trate (2 �M) at 10 ◦C (blue �). Data are also shown for a longer (32 bp) G·T substrate0.5 �M) collected with 5 �M TDG (red ♦). (For interpretation of the references toolor in this figure legend, the reader is referred to the web version of the article.)

nvolves effects on nonspecific binding (KES) and nucleotide flippingKflip), as discussed below.

.6. Temperature dependence of catalytic activity

We determined the temperature dependence of TDG catalyticctivity, and find striking and unexpected differences between G·Und G·T substrates. As shown in Fig. 4, catalytic activity increasesith temperature from 5 ◦C to 37 ◦C, with much steeper temper-

ture dependence for G·T relative to G·U substrates. Indeed, G·Tctivity increases 11-fold from 15 ◦C to 37 ◦C, compared to a 3-foldncrease for G·U activity. Notably, G·T activity is 5-fold higher at7 ◦C relative to 22 ◦C, the two temperatures used most frequently

n studies of TDG. These findings suggest important differencesn recognition and processing of G·T relative to G·U substrates, asiscussed below. Remarkably, two phases are observed in the tem-erature dependence for G·T activity; the phase discussed abovefrom 15 to 37 ◦C) and an even steeper phase from 5 to 15 ◦C.dentical results are obtained at 5 ◦C and 10 ◦C when the experi-

ents are repeated with 4-fold higher concentrations of enzymend substrate, indicating saturating enzyme conditions. In addi-ion, the same temperature dependence is observed for a longer·T substrate. Thus, the sharp decrease in G·T activity observed for

emperatures below 15 ◦C is due to an effect on nucleotide flippingnd/or the chemical step, rather than association of enzyme andubstrate. The potential mechanistic implications of these remark-ble findings are discussed below.

.7. TDG is unstable at 37 ◦C in the absence of DNA

Knowledge of the thermal stability of a protein is clearly impor-ant for experimental design, but such studies had not beeneported for TDG. While many previous kinetics and binding stud-es were conducted at 37 ◦C, the stability of TDG at 37 ◦C had noteen clearly established. To examine the thermal stability of TDG,

e performed a melting experiment using circular dichroism (CD)

pectroscopy, monitoring ellipticity at 222 nm (CD222). As shownn Fig. 5A, the CD melting curve indicates TDG is stable up to about5 ◦C, begins to unfold above 25 ◦C, and largely unfolds from 30 ◦C to

air 10 (2011) 545–553

45 ◦C. As discussed below, folding is irreversible, precluding a ther-modynamic analysis (including a meaningful Tm determination).

To assess whether unfolding leads to aggregation, we simul-taneously monitored CD222 and the photomultiplier (PM) voltage(Fig. 5B); the later responds to changes in light absorption and/orscattering [45]. For proteins that undergo reversible unfolding, nosubstantial change in PM voltage is expected, because the extinc-tion coefficient is not significantly temperature dependent andabsorption therefore remains relatively constant. In contrast, ifunfolding leads rapidly to aggregation (turbidity), an increase inPM voltage is expected due to light scattering [45]. As shown inFig. 5B, turbidity increases simultaneously with CD222 from 30 ◦Cto 35 ◦C, indicating unfolding coupled to aggregation. The drop inPM voltage above 36 ◦C is attributed to precipitation of aggregatedprotein. Dynamic light scattering experiments (Supplementary Fig.6) confirm that TDG aggregates at 37 ◦C, and show that aggregationis irreversible. In contrast, light scattering experiments show TDGis stable for at least 5 h at 22 ◦C.

We also examined the stability of TDG at 37 ◦C using Trp flu-orescence of TDG, as shown in Fig. 5C. Rapid emission scanswere collected following dilution of TDG into buffer at 37 ◦C.The spectrum collected immediately after dilution (15 s) reflectsthe heat-induced conformational change, with little aggregation.Compared to the spectra at 22 ◦C, a small increase in �max isobserved, suggesting the conformational change does not substan-tially increase solvent exposure of Trp160 or Trp383. At longertime points, �max remains constant and Imax increases, which isattributed to light scattering caused by aggregation, consistent withthe results from CD and dynamic light scattering experiments. Thechange in Imax is largely complete in 3 min, and fitting the timedependence of Imax to an exponential equation indicates aggrega-tion occurs with at rate constant of about 1 min−1 at 37 ◦C (for a1 �M concentration of TDG).

Observation of robust TDG catalytic activity at 37 ◦C (Fig. 4) indi-cates that substrate binding stabilizes TDG against heat-inducedunfolding. We used Trp fluorescence to examine the effect of DNAon the stability of TDG at 37 ◦C. As shown in Fig. 5D, a saturatingconcentration of G·TF substrate analog stabilizes TDG for over 2 h at37 ◦C, consistent with the tight binding of TDG to this DNA. TDG isalso stabilized at 37 ◦C by DNA containing a G·U mispair or an aba-sic site (not shown), consistent with even tighter binding to theselesions relative to G·T mispairs [26,28]. In addition, a saturatingamount of nonspecific DNA stabilizes TDG for over 2 h at 37 ◦C (notshown), consistent with our previous findings of remarkably tightbinding to nonspecific DNA (Kd = 0.3 �M) [28].

4. Discussion

4.1. pH dependence of catalysis and implications for themechanism of TDG

Determining the pH dependence of catalysis can be impor-tant for understanding enzymatic reactions, but such studies hadnot been reported for a TDG–MUG family member. For the G·Usubstrate, TDG exhibits relatively constant activity for pH 5.5–9,sharply decreasing activity for pH 9–10, and no activity at pH5 or 10.5. The absence of G·U activity at pH 5 and 10.5 can beexplained by inactivating structural changes for TDG at these pHvalues (Fig. 2). The sharp drop in activity for pH 9–10 is likelydue in part to severely diminished substrate binding at high pH

include deprotonation of uracil N3 (pKa = ∼9.5) [6,53] because thedU anion is expected to be a poor substrate [54,55], and/or struc-tural perturbation of TDG, which might be indicated by modestchanges in Trp fluorescence for pH 9–10 (Fig. 2). The pH profile

A. Maiti, A.C. Drohat / DNA Repair 10 (2011) 545–553 551

Fig. 5. Thermal stability of TDG monitored by circular dichroism (CD) and fluorescence spectroscopy. (A) Effect of temperature on the structural integrity of TDG (2.5 �M)monitored by CD at 222 nm (CD222). The increase in CD222 with temperature reflects heat-induced loss of secondary structure. As discussed in the text, thermal unfoldingof TDG is irreversible, precluding a thermodynamic analysis (including Tm determination). (B) In addition to CD222, the spectropolarimeter simultaneously records thephotomultiplier (PM) voltage, which increases linearly with decreases in light intensity caused by absorption or light scattering. Because a temperature-dependent change inabsorption is not expected (constant wavelength), changes in PM voltage are attributed to light scattering (turbidity) [44,45]. The initial increase in PM voltage is attributedto heat-induced aggregation of TDG, and the subsequent decrease is attributed to precipitation of aggregated protein. (C) Trp fluorescence spectra for TDG (1 �M) incubatedat 37 ◦C for various lengths of time (as shown in key). Also shown is the spectrum for TDG incubated at 22 ◦C (dotted line), which does not change over time (for >2 h). Thei atterina �M) ac se DN

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ncrease in fluorescence intensity with time for TDG at 37 ◦C is attributed to light scnd occurs with a rate constant of 1 min−1. (D) Trp fluorescence spectra of TDG (1ollected after incubation times of 1 min or 120 min. Our previous studies show the

btained for the 16 bp substrate and a fixed (2.5 �M) TDG concen-ration suggests greater sensitivity to increasing pH for G·T relativeo G·U substrates. However, the loss of G·T activity at pH 9 is almostully recovered by using a higher TDG concentration, such that theH profiles are roughly similar for G·T and G·U substrates for pH–9. The absence of G·T activity for pH > 9 likely reflects very largeffects of increasing pH on substrate binding. Ionization of the tar-et thymine (pKN3

a = ∼9.9) [6,53] might be a contributing factor.Our results provide no evidence for the requirement of a pro-

onated side chain in catalysis (i.e., a general acid), unless it has aKa > 9, nor evidence for an essential unprotonated group, unless

t has a pKa < 5. This result supports previous studies suggest-ng that a strictly conserved Asn is the only residue likely to bessential for catalysis in the TDG–MUG family of enzymes (N140or human TDG) [26,34,38,49,56]. Notably, UNG enzymes have anssential Asp in the corresponding position, which serves to acti-ate a water molecule for nucleophilic attack and/or stabilize thehemical transition-state of the reaction [54,55]. For E. coli UNG,his Asp has a pKa = 6.2, and catalytic activity is 10-fold lower atH 5.5 relative to pH 7.5 [31]. Our finding that TDG retains full cat-lytic activity at pH 5.5 underscores the key differences in catalyticechanism between members of the UNG enzyme superfamily.

.2. Effects of increasing ionic strength on TDG activity

The catalytic activity of TDG (kobs) decreases sharply withncreasing ionic strength, particularly for G·T substrates, and thisan be attributed in part to deleterious effects on nucleotide flippingKflip) or the chemical step (kchem) of the TDG reaction [6,10,26]. Wend a 4-fold loss in kobs for G·T activity as ionic strength increases

g arising from TDG aggregation. The intensity change is largely complete in 3 min,t 37 ◦C, in the presence of G·TF analog (2 �M) or nonspecific DNA (NS28, 10 �M),A concentrations are saturating for these [28].

from 0.06 M to 0.16 M. Observation that activity is not recovered byusing a higher enzyme concentration at 0.16 M ionic strength indi-cates saturating enzyme conditions, such that the decrease in kobsis not due to an effect on enzyme–substrate association (i.e., forma-tion of the initial E·S complex). Much larger decreases in kobs areobserved at higher ionic strength, although this is likely due in partto effects on enzyme–substrate association. Although increasingionic strength can be expected to weaken nonspecific protein–DNAinteractions, adverse effects on Kflip or kchem for a DNA glycosy-lase reaction are not necessarily expected. Indeed, studies of AAGshow relatively constant single-turnover activity as ionic strengthincreases up to 0.3 M [57]. In sharp contrast, TDG exhibits a 100-fold decrease in single turnover activity (G·T) at 0.3 M relative to0.06 M ionic strength.

Deleterious effects of increasing ionic strength on nucleotideflipping could potentially involve disruption of electrostatic inter-actions formed with the flipped base in the active-site, orinteractions involving Arg275, a side chain that is important for sta-bilizing nucleotide flipping, particularly for G·T substrates [26,38].Increasing ionic strength could potentially slow the chemical stepby perturbing electrostatic interactions that stabilize the chemicaltransition state. The greater effect of ionic strength on kobs for G·Tversus G·U suggests a larger adverse effect on Kflip for G·T process-ing, consistent with our previous findings that nucleotide flippingis inherently less stable for dT relative to dU nucleotides [26,28,38],

or perhaps a larger adverse effect on kchem for G·T versus G·U sub-strates.

In addition to the effects of increasing ionic strength on Kflipand/or kchem indicated by the results above, the equilibrium bindingstudies reveal a large adverse effect on nonspecific DNA binding.

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52 A. Maiti, A.C. Drohat / D

his is indicated by the 45-fold decrease in G·T binding affinity asonic strength increases from 0.06 M to 0.16 M, and likely involveserturbation of contacts with DNA backbone phosphates, amongther potential interactions. Notably, our findings reveal conditionsf ionic strength (0.06 M) that allow for measurement of a wideange of substrate binding affinities, which could be important fortructure–activity studies to determination the effect of enzymeutations or substrate modifications on substrate binding affinity

or TDG.

.3. Effects of temperature on catalytic activity and enzymetability

While many previous studies of TDG were performed at physio-ogical temperature, to our knowledge, it had not been established

hether TDG is stable at 37 ◦C. We find TDG aggregates rapidlynd irreversibly at 37 ◦C, although it can be stabilized by a suffi-ient quantity of specific or nonspecific DNA. These results suggestaution when performing studies at 37 ◦C, to ensure TDG remainsully active for the duration of an experiment. Provided such con-itions can be identified, the 5-fold higher activity of TDG at 37 ◦Cersus 22 ◦C (for G·T substrates) may be experimentally useful, andield results that are more physiologically relevant. Alternatively,e show that TDG is stable for several hours at 22 ◦C in the absence

f stabilizing DNA.TDG catalytic activity (kobs) increases with temperature

5–37 ◦C) for G·T and G·U substrates, and the temperature depen-ence is much steeper for G·T activity. This key observationas unexpected, and suggests a dramatic difference in catal-

sis for these closely related substrates, which is consideredelow. We first note that for non-enzymatic N-glycosylic bondleavage of dU and dT nucleosides, the thermodynamic param-ters (�H‡, T �S‡) are highly similar [29,58]. The reactions areverwhelmingly enthalpic with a small entropic contribution�H‡ = 28.6 kcal/mol, T �S‡ = −1.9 kcal/mol for dU, 25 ◦C), consis-ent with a highly dissociative mechanism [29,58,59]. Similarly,nzymatic N-glycosylic bond cleavage by UNG and MutY involvesstepwise mechanism [59–61]. This precedent and our previous

tudies suggest the TDG reaction is highly dissociative, perhapstepwise [6,55], and activation parameters for the chemical step�H‡, T �S‡) seem unlikely to differ dramatically for G·T and G·Uubstrates. Thermodynamic parameters for TDG catalysis (kobs)an be obtained from an Arrhenius plot (Supplementary Fig. 7),hich gives �H = 18.5 ± 1.0 kcal/mol, T �S = −2.5 ± 1.0 kcal/mol

or G·T activity (for T ≥ 15 ◦C), andH = 8.5 ± 0.6 kcal/mol, T �S = −10.8 ± 0.6 kcal/mol for G·U activ-

ty. Because these parameters are derived from kobs, they cannclude contributions from nucleotide flipping or an associatedDG conformational change (and are not shown as �H‡, T �S‡).ne interpretation is that for G·T catalysis, nucleotide flipping

s in rapid equilibrium and does not contribute substantially toobs, consistent with thermodynamic parameters that might bexpected if kobs is dominated by kchem (i.e., small T �S‡). For G·Uatalysis, reverse nucleotide flipping could be much slower (dueo favorable active-site interactions) such that nucleotide flippingontributes to kobs, as suggested by a large entropic component for·U catalysis. Our recent observations indicate reverse nucleotideipping is indeed much faster than kchem for G·T substrates andomparable to kchem for G·U substrates (Fitzgerald ME and DrohatC, manuscript in preparation). This could be due to steric effectsith T (but not U) that shorten the lifetime of the flipped state. As

iscussed above (Section 1), our previous studies strongly suggesthat steric effects weaken substrate binding and catalytic activitynd for T and other bases with a bulky substituent at the C-5osition (5-BrU, 5-iodoU, etc.) [6,10,26,28]. Steric effects with Tight be needed to minimize excision of T from A·T pairs, and may

[

air 10 (2011) 545–553

account for the relatively weak G·T activity of TDG, which mustbalance the needs for efficient damage repair and avoidance ofaberrant activity on undamaged DNA.

Another remarkable finding is the sharp transition in tempera-ture dependence of catalysis (∼15 ◦C) for G·T but not G·U substrates.Similar observations were reported for other enzymes, and in somecases the transition was correlated to an enzyme conformationalchange [62]. Previous studies also find that a transition is observedfor some but not all substrates. It seems plausible that the transitioncould reflect a TDG conformational change that effects G·T but notG·U activity. The very steep temperature dependence of G·T catal-ysis for T < 15 ◦C may reflect a conformation of TDG in which stericeffects strongly suppress G·T (but not G·U) activity, such that activ-ity is limited by an enzyme conformational change that precedescatalysis. Of course, other factors could contribute, and additionalstructural and biochemical studies are needed to further explorethese remarkable findings.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

The CD melting data were collected by Leslie Eisele at the Bio-chemistry Core facility of the Wadsworth Center, NY State Dept.of Health. This work was supported by a grant from the NationalInstitutes of Health (R01-GM-072711), and the Greenebaum Can-cer Center, School of Medicine, University of Maryland Baltimore.We thank the reviewers for helpful suggestions.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.dnarep.2011.03.004.

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