Chromatographic isolation of the functionally active MutS protein covalently linked to deoxyribonucleic acid

Journal of Chromatography A, 1389 (2015) 19–27

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Chromatographic isolation of the functionally active MutS protein covalently linked to deoxyribonucleic acid夽 Mayya Monakhova a , Alexandra Ryazanova a , Andreas Hentschel b,1 , Mikhail Viryasov a,∗ , Tatiana Oretskaya a , Peter Friedhoff b , Elena Kubareva a a Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia b Institute for Biochemistry, FB 08, Justus Liebig University, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany

a r t i c l e

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Article history: Received 11 December 2014 Received in revised form 10 February 2015 Accepted 16 February 2015 Available online 21 February 2015 Keywords: Mismatch repair MutS protein DNA–protein crosslinking Anion exchange chromatography

a b s t r a c t DNA metabolism is based on formation of different DNA–protein complexes which can adopt various conformations. To characterize functioning of such complexes, one needs a solution-based technique which allows fixing a complex in a certain transient conformation. The crosslinking approach is a popular tool for such studies. However, it is under debate if the protein components retain their natural activities in the resulting crosslinked complexes. In the present work we demonstrate the possibility of obtaining pure DNA conjugate with functionally active protein using as example MutS protein from Escherichia coli mismatch repair system. A conjugate of a chemically modified mismatch-containing DNA duplex with MutS is fixed by thiol–disulfide exchange reaction. To perform a reliable test of the protein activity in the conjugate, such conjugate must be thoroughly separated from the uncrosslinked protein and DNA prior to the test. In the present work, we employ anion exchange chromatography for this purpose for the first time and demonstrate this technique to be optimal for the conjugate purification. The activity test is a FRET-based detection of DNA unbending. We show experimentally that MutS in the conjugate retains its ability to unbend DNA in response to ATP addition and find out for the first time that the DNA unbending rate increases with increasing ATP concentration. Since the crosslinked complexes contain active MutS protein, they can be used in further experiments to investigate MutS interactions with other proteins of the mismatch repair system. © 2015 Elsevier B.V. All rights reserved.

1. Introduction One could hardly overestimate the importance of the studies concerning structure and conformational changes in DNA–protein complexes. Among all the known structures of DNA–protein complexes, most have been determined by X-ray crystallography. Therefore, they represent the static models of proteins covalently or non-covalently bound to relatively short DNA fragments [1–8]. However, many metabolic processes are based on formation of large macromolecular complexes which change their

夽 Presented at the 30th International Symposium on Chromatography (ISC 2014), Salzburg, Austria, 14–18 September 2014. ∗ Corresponding author at: Chemistry Department, M.V. Lomonosov Moscow State University, Leninskie Gory 1/3, Moscow, Russia. Tel.: +7 916124 72 15; fax: +7 499196 29 37. E-mail address: [email protected] (M. Viryasov). 1 Present address: Leibniz-Institut für Analytische Wissenschaften, Otto-HahnStr. 6b, D-44227 Dortmund, Germany. http://dx.doi.org/10.1016/j.chroma.2015.02.045 0021-9673/© 2015 Elsevier B.V. All rights reserved.

conformations during functioning. Investigating such complexes requires studying transient intermediates and hence demands a solution-based technique which would allow fixing a complex in a certain conformation. Crosslinking is a popular tool for this purpose. We mention below some findings obtained for mismatch repair system (MMR) using this technique. The MMR system maintains genome stability by correcting noncanonical nucleotide pairs (mismatches) and small loops which arise in cellular DNA. Main MMR steps are: (1) mismatch searching and recognition, (2) DNA strand discrimination and cleavage, (3) mismatch removal, (4) restoring DNA integrity [9]. The major active components of this system in Escherichia coli are MutS, MutL, and MutH proteins. At the first stage of the MMR process, MutS searches for a mismatch, binds it and bends the DNA forming the so-called recognition clamp [10–12]. When bound to a mismatch, MutS loses its affinity to ADP and binds ATP thereby being activated to form a stable sliding clamp. MutL binds the MutS–DNA complex and acts as a mediator activating the strand-discrimination endonuclease MutH. MutH binds at a hemimethylated

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5 -Gm6 ATC-3 /3 -CTAG-5 site (where m6 A is 6-methyl-2 deoxyadenosine residue) and nicks the non-modified daughter DNA strand starting a cascade of events which involves DNA helicase II (UvrD), one of several exonucleases, DNA polymerase III, and DNA ligase [9]. Since MutS and MutL have complex and very mobile structures, the crosslinking technique proved to be useful for studying these proteins. MutL is a homodimer where each subunit consists of two domains separated by a long flexible linker. Although the structures of both MutL domains were successfully determined by X-ray crystallography [13,14], full-length MutL is not crystallized so far, not to mention larger complexes which MutL forms with other components of the MMR system. However, many details of their structures were clarified using protein–protein crosslinking: physical interaction between the N-terminal ATPase domain and the C-terminal endonuclease domain of MutL was found [15]; interaction site between MutL and MutH was mapped [16,17]; the mismatch-binding and connector domains of MutS were shown to be in vicinity of MutL N-terminal domain [18]. As to MutS, this protein consists of many domains. The recognition clamp and the sliding clamp states of MutS are expected to differ significantly from each other. Large conformational changes in DNA and the mismatch-binding domain have been demonstrated [19–21]. MutS conformation after an ATP-induced conformational change was successfully fixed by intramolecular crosslinking [18]. MutS from E. coli exists in an equilibrium between dimers and tetramers which complicates its biophysical characterization [22]. In order to stabilize tetramers, protein–protein crosslinking was used; the crosslinked tetramers were separated from dimers by size-exclusion chromatography, mixed with their target DNA, and studied using small-angle X-ray scattering [23]. Conformational mobility of MutS–DNA complexes complicates their characterization and many details of their functioning still remain unclear. Obtaining a covalently fixed MutS–DNA complex would be extremely useful for detalization of MutS with DNA as well as for investigating interactions of this complex with MutL and MutH. Yet, a proof is necessary that the crosslinked complex retains its natural structure and activity in order to get biologically reliable results. Crosslinking combined with functional studies of protein–DNA complexes requires the following steps: (1) regioselective reaction between DNA and protein; (2) purification of the conjugate; (3) confirmation of the functional activity of the purified complex. Here we used the approach for the protein–DNA crosslinking similar to that described by Verdine and Norman [24]. It consists of making a variant of the DNA-binding protein which contains a single cysteine residue in a position suitable for crosslinking and performing the thiol–disulfide exchange reaction between this cysteine and a disulfide group inserted in DNA [24–29]. Previously, we applied this protein–DNA crosslinking strategy for trapping MutS on a 29-bp DNA which carried the reactive group on the 3 -end [30]. In the present work we combine DNA–protein crosslinking with chromatographic purification to obtain pure DNA–protein complex and prove its functional activity by fluorescence resonance energy transfer (FRET) and stopped-flow kinetic analysis. The new strategy is tested using MutS protein from E. coli MMR system and its substrate, a mismatch-containing 59-bp DNA duplex which carries a reactive group and two fluorophores. Our protocol permits obtaining large amounts of highly pure MutS–DNA conjugate which retains its activity and therefore can be used in further experiments to study conformational rearrangements during the MMR initiation.

2. Materials and methods All chemicals used in this work were of molecular biology grade purity, unless stated otherwise. 2.1. DNA Oligonucleotides without fluorescent labels (HPLC grade) were synthesized by Eurogentec (Seraing, Belgium). Oligonucleotides with fluorescent labels (PAGE grade) were ordered from IBA (Göttingen, Germany). DNA duplexes were prepared by annealing complementary DNA strands in water; the strand without fluorophores was taken in a slight excess (5–10%) over that with fluorophores. 2.2. Crosslinkers The following four crosslinkers were used: N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), N-[(succinimidyl)oxy]-15oxo-3,6,9,12-tetraoxapentadecyl-3-(2-pyridyldithio)propanamide (PEG4 -SPDP), N-[(succinimidyl)oxy]-27-oxo-3,6,9,12,15,18,21,24octaoxaheptacosyl-3-(2-pyridyldithio)propanamide (PEG8 -SPDP), and N-[(succinimidyl)oxy]-39-oxo-3,6,9,12,15,18,21,24,27,30,33, 36-dodecaoxanonatriacontyl-3-(2-pyridyldithio)propanamide (PEG12 -SPDP). SPDP, PEG4 -SPDP and PEG12 -SPDP were from Thermo Scientific (Rockford, AL, USA), while PEG8 -SPDP was from Celares (Berlin, Germany). 2.3. DNA modification An oligonucleotide containing a C5-aliphatic amino group coupled to T through a hexamethyleneacrylamide linker (4 nmol) was dissolved in 45 ␮l of 100 mM sodium borate buffer (pH 8.5). Each crosslinker was dissolved in DMSO to get the 500 mM solution, aliquoted and stored in a bag with desiccants at −20 ◦ C. The oligonucleotide solution was mixed with 5 ␮l of the crosslinker solution. The resulting mixture was well stirred and left at room temperature overnight. 2.4. Purification of the modified DNA The modified oligonucleotide was purified of the non-reacted crosslinker by size-exclusion chromatography using 0.5 ml Zeba spin desalting columns (Thermo Scientific, Rockford, AL, USA) with 7 K or 40 K molecular weight cut-off (MWCO) resin. Deionized water was eluent. For controlling the crosslinker removal efficiency, 4 ␮l of 1 mM 2-mercaptoethanol was added to 1 ␮l of the reaction mixture and kept for 15 min at room temperature. Then optical density of the sample was measured at NanoDrop spectrophotometer (Thermo Scientific, Rockford, AL, USA) and amount of the released pyridine-2-thione was assessed via its absorption at 343 nm. To achieve the desired purity level (see Section 3.3), we repeated gel filtration over Zeba columns. The reaction efficiency was assessed by electrophoresis in native 20% polyacrylamide gel (10 cm × 8 cm × 0.1 cm, 15 mA for 2 h in 89 mM Tris–borate, 1 mM EDTA, pH 8.3) followed by ethidium bromide staining. The dyes bromophenol blue and xylene cyanol were used as markers during electrophoresis. 2.5. Protein expression and purification MutS variant was expressed and purified as described earlier [10,23,31] and was kindly provided by Prof. T. Sixma (Division of Biochemistry, Netherlands Cancer Institute). The protein was

M. Monakhova et al. / J. Chromatogr. A 1389 (2015) 19–27

stored in 10 mM HEPES/KOH (pH 7.9), 200 mM KCl, 1 mM EDTA and 10% (w/v) glycerol at −80 ◦ C. 2.6. MutS crosslinking to DNA The crosslinking was performed by adding the modified DNA duplex (1 ␮M) to the protein (4 ␮M per monomer) pre-incubated in buffer A (25 mM HEPES/KOH, pH 7.5, 125 mM KCl, 5 mM MgCl2 ) with different amounts of ATP (0, 10, 100 or 1000 ␮M in different experiments). The mixture was kept for 30 min on ice or at 37 ◦ C. The crosslinking was performed in the volume of 10 ␮l (for analytical purposes), 300 or 600 ␮l (for the subsequent chromatographic purification). 2.7. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) SDS–PAGE under non-reducing conditions was used to analyze the crosslinked mixtures as well as the fractions obtained from ion exchange chromatography. The gels were casted in 10 cm × 8 cm × 0.1 cm glass plates. The separating gel contained 6% (w/v) acrylamide:N,N -methylenebisacrylamide mixture (29:1), 420 mM Tris–HCl, pH 8.8, 0.1% (w/v) SDS. The stacking gel contained 4% (w/v) acrylamide:N,N -methylenebisacrylamide mixture (29:1), 130 mM Tris–HCl, pH 6.8, 0.1% (w/v) SDS. The samples were diluted with 5× loading buffer (160 mM Tris–HCl, pH 6.8, 2% (w/v) SDS, 40% (w/v) glycerol, 0.01% (w/v) bromophenol blue) and then loaded onto gel without heating. Two protein molecular weight markers were used, SpectraTM Multicolor High Range (40–300 kDa) and PageRulerTM (10–170 kDa) (Thermo Scientific, Rockford, AL, USA). Electrophoresis was performed at 90 V for 10 min and then at 180 V for 50 min using the following buffer: 25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS, pH 8.5. Gels were stained with InstantBlue (Expedeon, Germany), a Coomassie-based protein staining solution. 2.8. Ion exchange HPLC HPLC experiments were performed using LaChrom Elite HPLC system (Hitachi, Tokyo, Japan) with EZChrom Elite software. A prepacked 1 ml column (bed dimensions 7 mm × 25 mm) was used, either HiTrap Q HP or HiTrap Capto Q (GE Healthcare Life Sciences, Little Chalfont, UK). The ion exchange columns were equilibrated with 500 mM KCl in buffer B (10 mM HEPES/KOH, pH 7.9, 10% (w/v) glycerol, 1 mM EDTA) at a flow rate of 0.5 ml/min. The crosslinking mixture was centrifuged for 5 min at 13,000 rpm using table top centrifuge to precipitate any unsoluble particles. These samples in buffer A were injected in the HPLC system. The injection volume was 600 ␮l for the mixture with DNA containing no fluorescent labels and only 300 ␮l for the mixture with double-labeled DNA. The chromatography separations were carried out at room temperature at a flow rate of 0.5 ml/min. After injection the column was washed isocratically for 20 min with 500 mM KCl in buffer B in order to remove ATP and protein. The DNA–protein conjugate and free DNA were eluted using a step salt gradient from 500 to 850 mM KCl in buffer B (20–35 min: 650 mM KCl, 35–40 min: linear increase from 650 to 750 mM KCl, 40–50 min: 750 mM KCl, 50–55 min: linear increase from 750 to 850 mM KCl, 55–65 min: 850 mM KCl). The absorbance of the eluate was registered at four different wavelengths, namely 260, 280, 490 and 590 nm (for DNA, protein, Atto-488 and Alexa-594 absorbance, respectively). The 500 ␮l fractions were collected and kept on ice. The fractions corresponding to each peak were analyzed by SDS–PAGE. Those containing the crosslinked complex were mixed together, aliquoted, frozen in liquid nitrogen and stored at −80 ◦ C. After eluting of the free DNA, the columns were washed with 1000 mM KCl in buffer B for 25 min to ensure total removal of any negatively charged substances. The

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columns were stored in 20% ethanol solution at room temperature. The yield of the DNA–MutS conjugate was estimated by integrating the peaks corresponding to the conjugate and to the free DNA. Standard deviation (SD) of the yield was calculated using three independent HPLC purifications. 2.9. Buffer exchange For any functional testing, an aliquot of the crosslinked complex was thawed and then the buffer was exchanged into buffer C (50 mM K-phosphate, pH 7.4, 50 mM KCl, 0.1 mM EDTA, 5 mM MgCl2 ) using Zeba 40K columns. The mixture in buffer C was kept on ice and used during 1–2 h. 2.10. Steady-state FRET and anisotropy assay The assay was performed at FluoroMax-4 Spectrofluorometer (Horiba Jobin Yvon, Kyoto, Japan) at room temperature. A quartz cuvette (Hellma, Müllheim, Germany) of 100 ␮l with an optical path of 10 mm was used. An aliquot of the crosslinked complex in buffer C was further diluted with buffer C to reach 100 ␮l. The fluorescence emission spectra were recorded from 500 to 800 nm using excitation wavelength of 470 nm. Then anisotropy of the donor fluorescence was measured using excitation wavelength of 470 nm and emission wavelength of 535 nm. The emission and excitation slits were 2 and 5 nm, respectively for the both measurements. Then ATP was added up to 1 mM followed by DTT addition up to 10 mM. Finally, proteinase K was added to destroy the protein completely. Emission spectra were recorded and anisotropy was measured after the addition of each component. Each experiment was repeated three times; the SD of the quantitative parameters did not exceed 5%. 2.11. Stopped-flow experiments Fast kinetics was measured at stopped-flow device SF-61SX2 (TgK Scientific, Bradford-on-Avon, UK) using excitation wavelength of 436 nm, filter ET525/50 M (Chroma Technology, Olching, Germany) for the donor fluorescence and a long pass filter OG590 (Schott, Mainz, Germany) for FRET. Eight thousand one hundred ninety-two data points were collected over logarithmic time scale. The first syringe contained 8 nM crosslinked complex in buffer C, the second syringe contained different concentrations of ATP (2, 20, 200, and 2000 ␮M) in buffer C. Thus, the final concentrations were twofold lower than those in the syringes. The kinetic curves for each concentration were recorded two or three times and were found to overlap almost perfectly; they were averaged and analyzed using Origin software 9.0. 3. Results and discussion 3.1. Design of the oligonucleotides MutS from E. coli was successfully crystallized in complex with a 30-bp DNA which contained the G/T pair demonstrating a characteristic kink of 60◦ at the mismatch [10]. The data from footprinting show that MutS covers ∼20 bp [32–34]. However, it is not completely clear what is the minimal DNA length necessary to detect a triple complex between MutS, MutL and DNA. Some data claim that DNA should be as short as 46 or 60 bp but not as short as 37 or 42 bp [18,34,35]. To create a system which would be useful for future investigations of the MMR proteins, we aimed at providing more space for such “additional” interactions and used a longer DNA duplex (59 bp). This duplex contained one G/T mismatch (Fig. 1A), since G/T is recognized by MutS with a high affinity [36]. Mismatched DNA bending during interaction with MutS as well as DNA

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Fig. 1. Oligonucleotides used in the present work and reactions which provide the DNA–protein crosslinking. (A) DNA duplex with a G/T mismatch in position 38. The “top” strand carries two fluorophores, Alexa-594 in position 25 and Atto-488 in position 48. The “bottom” strand carries a C5 aliphatic amino group linked to T in position 30 which can be modified with commercial crosslinkers SPDP, PEG4 -SPDP, PEG8 -SPDP or PEG12 -SPDP. The distance between C atom of the carbonyl group and S atom is shown for each crosslinker. (B) Modification of a primary amino group attached to a single-stranded DNA with SPDP reagent. One of the products of the first reaction, NHS, absorbs light at the same wavelength as DNA. Therefore, the modified DNA should be thoroughly purified from NHS before measuring the DNA concentration. One of the products of the second reaction, pyridine-2-thione, can be used for quantification of initial pyridyldithio groups in the reaction mixture. (C) Crosslinking reaction between double-stranded DNA carrying pyridyldithio group and thiol group of a protein molecule.

unbending in recognition complex in the ATP presence is thought to be the characteristic initial steps of MMR process [12,19,37–39]. Therefore, we considered MutS ability to bend and unbend DNA as the evidence of its activity. To detect easily such DNA deformations, we placed two fluorophores forming a FRET pair in one DNA strand, on the both sides of the mismatch (Fig. 1A). Atto-488 served as a fluorescence donor, while Alexa Fluor® 594 (Alexa-594) was an acceptor. The distance between these fluorophores was 22 bp ˚ somewhat exceeding the Förster radius of this FRET (about 75 A, pair). Each fluorophore was covalently attached to the position 5 of a thymine heterocyclic base. The fluorophores linked this way to heterocyclic bases do not impair MutS binding to a mismatched base pair [19]. To perform subsequently crosslinking between DNA and protein, we placed an aliphatic amino group attached to the position 5 of a thymine heterocyclic base through a C6 linker. The amino group was modified by different reagents carrying pyridyldithio group (Fig. 1A) which is able to react with the protein cysteine residue (see below). The position for this modification was chosen on the basis of our previous results where a reactive group (a commercially available thiol modifier HO(CH2 )3 SS(CH2 )3 – attached to the DNA 3 -end) placed 7 bp away from the mismatch was used successfully for DNA crosslinking to single-cystein MutS variant, MutS(N497C/801–853) [30]. The linker length can be critical for crosslinking reaction as a too short linker could provide a low yield of the conjugate. Moreover, protein in such conjugate could happen to lack its natural activity. On the other hand, a too long linker

could result in a crosslinked complex where the components move freely in relation to each other and thus do not show the properties of their native complex. Therefore, we tried four crosslinking reagents to obtain DNA with four linkers of different length, from 6.8 to 54.1 A˚ (Fig. 1A). In contrast to the previous FRET-based studies of the MutSinduced DNA bending [19,37,40], we had both fluorophores attached to the same DNA strand. The other DNA strand contained only one modification, the aliphatic amino group. This strand was modified with the crosslinkers. Thus, it was possible to introduce different reactive groups in the DNA duplex without changing the strand containing the FRET pair. 3.2. Choice of the MutS mutant We used a protein variant derived from the full-length cysteinefree MutS which carried two additional point mutations, N497C and D835R. We will refer to this mutant as MutS-497 throughout this article for the sake of brevity. This protein has molecular weight of 95 kDa. Wild-type MutS exists in equilibrium between dimers and tetramers in E. coli cells [22]. The presence of tetramers complicates significantly analysis of experimental results, while the D835R mutation results in a charge inversion and thus prevents MutS tetramerization without disruption of MutS dimers [41]. The Cys497 is located in the clamp domain and is close to the bound DNA. This MutS variant was successfully used in our previous crosslinking experiments [30].

M. Monakhova et al. / J. Chromatogr. A 1389 (2015) 19–27

Fig. 2. Modification of the single-stranded 59-mer DNA which carried a primary amino group coupled to a thymine heterocyclic base through a C6 linker (59-T-NH2 ) with commercial crosslinkers (SPDP, PEG4 -SPDP, PEG8 -SPDP or PEG12 -SPDP). The reaction products were analyzed in native 20% PAGE followed by ethidium bromide staining. The modified DNAs had decreased electrophoretic mobilities in comparison with the initial DNA (59-T-NH2 ). The longer was the crosslinker, the bigger was the electrophoretic mobility shift. The lanes with the modified DNA contained no trace of the initial DNA and thus we considered the reaction to be quantitative. The dyes bromophenol blue and xylene cyanol were used as markers during PAGE; they were washed off during ethidium bromide staining and therefore are not shown in this figure.

3.3. DNA modification with crosslinkers Conjugation of SPDP reagents to primary amines via the N-hydroxysuccinimide (NHS) ester (Fig. 1B) is a well-known highyield reaction [42]. The modified DNA demonstrated a slight decrease of electrophoretic mobility which was detectable even in a native polyacrylamide gel (without urea addition), as demonstrated in Fig. 2. No trace of the initial DNA was visible in the lanes which contained the DNA with pyridyldithio group (Fig. 2). Thus, the reaction was considered quantitative for each one of the four crosslinkers. NHS group is known to absorb light strongly at 260 nm (ε260 = 9700 M−1 cm−1 ) [42]. As this maximum coincides with the maximum of DNA absorbance, the DNA concentration can be determined spectrophotometrically only after a thorough removal of the crosslinker excess. Zeba spin desalting columns were used for this purpose. Since the crosslinker-modified oligonucleotide has a molecular weight of 19 kDa, Zeba columns with a 7 kDa MWCO would be recommended for purifying such DNA from the nonreacted crosslinker. However, due to the rod-shaped geometry of DNA, the crosslinker-modified oligonucleotide was found to be eluted with a high yield even from Zeba columns with MWCO of 40 kDa. The reaction between pyridyldithio group of the modified DNA or non-reacted crosslinkers and sulfhydryl group of 2-mercaptoethanol (Fig. 1B) results in pyridine-2-thione displacement [43]. This fact was used to estimate the amount of the crosslinker remained in the mixture. An aliquot was mixed with 2-mercaptoethanol and concentration of the released pyridine-2-thione was determined by measuring the absorbance at 343 nm (ε343 = 8080 M−1 cm−1 ). For a perfectly pure modified DNA, the amount of pyridine-2-thione would be equal to the DNA amount calculated from the absorbance at 260 nm (ε260 = 575,700 M−1 cm−1 for the oligonucleotide with the aliphatic amino group). Several sequential runs using Zeba columns were required to obtain the desirable purity, depending on the crosslinker length (typically, two repetitions for SPDP, three

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Fig. 3. MutS-497 crosslinking to 59-bp DNA duplexes, each one containing a mismatch and carrying a pyridyldithio group coupled to a heterocyclic base through a linker of a certain length (see Fig. 1). The reaction products were analyzed by 6% SDS–PAGE under non-reducing conditions followed by Coomassie blue staining. Each variant of the modified DNA duplexes yielded a DNA–protein conjugate, and the smallest yield was in the case of the shortest linker (59-SPDP). The right lane represents a control experiment where no DNA was added to the protein. The left lane contains protein molecular weight markers; their apparent molecular weight (in kDa) is shown.

repetitions for PEG4 -SPDP, four repetitions for PEG8 -SPDP, and five repetitions for PEG12 -SPDP). 3.4. MutS-497 crosslinking to DNA Each DNA duplex carrying pyridyldithio group was successfully crosslinked to MutS-497. Formation of the crosslinked complex was detected by SDS–PAGE (Fig. 3). Although MutS-497 functions as homodimer, only one MutS subunit can be crosslinked to the modified DNA (Fig. 1 C). In the SDS–PAGE under non-reducing conditions, the crosslinked complex dissociates producing a free protein subunit and a subunit covalently linked to the DNA (Fig. 3). At first, we performed the crosslinking reactions on analytical scale (reaction volume was 10 ␮l) trying to increase the yield of the DNA–protein conjugate. For this purpose, a qualitative assessment of the bands in SDS-gel was sufficient. For a more effective reaction one of components should be taken in excess. We considered the modified DNA to be a more costly reagent than the non-modified protein and therefore used the protein excess over DNA in order to maximize the crosslinking yield. The twofold excess (calculated per MutS dimer) was already enough; a larger excess did not increase the conjugate yield anymore. The reaction mixture was kept for 30 min at 0 ◦ C or at 37 ◦ C. Surprisingly, the yield was slightly higher in the former case. Most likely, a fraction of the protein was inactivated during the incubation at 37 ◦ C. We tried also to use different concentrations of ATP or ADP in reaction mixture, namely 0, 10, 100, and 1000 ␮M. For the MutS-497 mutant, the crosslinking efficiency did not change depending on the type or concentration of the nucleotide (data not shown). One can see from the gel (Fig. 3) that the amount of the crosslinked complex was similar in case of PEG4 -SPDP, PEG8 -SPDP and PEG12 -SPDP crosslinkers. Since we found no significant differences between these three variants, we used only PEG4 -SPDP and PEG12 -SPDP for the future experiments. The shortest linker in the SPDP reagent indeed resulted in the lowest yield of the conjugate (Fig. 3). Some amount of MutS dimers (Fig. 3, the highest band in all lanes) was always present in the MutS-497 preparation due to

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the covalent bond formation between the Cys497 residues in the subunits of the MutS homodimer. This bond could be destroyed by adding DTT excess (data not shown). Since DTT destroyed also pyridyldithio group, it was never present in our crosslinking mixtures. However, considering the twofold protein excess over DNA, some amount of MutS dimers did not decrease the crosslinking yield. An unusual position of this band on a gel (about 300 kDa) seems too high for MutS dimer (190 kDa). However, electrophoretic mobility of a crosslinked polymer can differ significantly depending on the position of the crosslink in the molecule [44]. 3.5. HPLC purification of the crosslinked complex In order to preserve a functionally active complex, we searched for appropriate purification conditions that should provide the following: (1) the stability of MutS homodimeric status; (2) preserving the DNA-binding and ATPase activity of MutS; (3) maintaining the double helix DNA structure. Previously, a crosslinked complex of MutS and a 29-bp DNA was purified via size-exclusion chromatography on a Superdex-200 10/300 GL column (GE Healthcare Life Sciences, Little Chalfont, UK) [30]. The first eluted peak contained MutS dimer crosslinked to DNA with a slight impurity of noncrosslinked MutS dimer, while the second peak contained free DNA. However, we failed to resolve these peaks when the experiment was repeated with a 59-bp DNA. Thus, the central point of the present work was developing another chromatographic method for the crosslinked complex purification. Since DNA is a large polyanion, we tried to use anion exchange chromatography. Two prepacked columns of the HiTrap series were tested, with Capto Q and with Q HP resin. Both resins are strong quaternary ammonium anion exchangers; they are coupled to different matrices, highly crosslinked agarose with dextran surface extender in the case of Capto Q and 6% crosslinked agarose in the case of Q HP. We found no difference between these resins for our application and further used the Q HP column. As preliminary experiments, we performed chromatography with a linear salt gradient (100–1000 mM KCl) for the MutS protein alone, for the nonmodified DNA duplex alone and for the reaction mixture containing the DNA–protein conjugate. Taking into account the obtained elution times, we developed the following step gradient. The column was pre-equilibrated with 500 mM KCl in buffer B. The sample was injected and the salt concentration was kept constant for 20 min to ensure binding of the DNA-containing components and washing away the non-crosslinked protein. Indeed, the flowthrough (a large peak at 3–5 min) contained the non-reacted protein together with ATP and ADP molecules. Then 650 mM KCl was applied for 15 min to provide full elution of the target peak, i.e., the crosslinked complex. Further, the salt concentration was increased up to 750 mM and kept constant for 10 min to obtain the last peak which contained the free DNA. The fractions corresponding to each peak were collected and analyzed by SDS–PAGE with Coomassie blue staining. The anion exchange protocol was developed initially for nonlabeled DNA duplexes crosslinked to MutS. The elution process was monitored at two wavelengths (260 nm for DNA and 280 nm for protein; Fig. 4). After obtaining good peak resolution, we switched to similar reaction mixtures where DNA duplexes were doublelabeled. The chromatogram was recorded at four wavelengths (adding 490 nm for Atto-488 and 590 nm for Alexa-594; Fig. 5A and B). The analysis of eluted fractions corresponding to the MutS–DNA conjugate peak by SDS–PAGE is presented in Fig. 5C. We have found that MutS frozen in a buffer without glycerol retained absolutely no activity after thawing. Therefore, we used 10% glycerol in any buffer for storage of the protein or the crosslinked complex. The eluent for ion exchange chromatography also contained 10% glycerol in order to freeze the eluted fractions as fast as possible. In control experiment, we performed ion exchange

Fig. 4. Separation of reaction mixtures containing MutS-497 conjugates with different unlabeled DNA duplexes by ion exchange chromatography with detection at 260 nm. (A) The DNA duplex is 59-SPDP. The retention times are 28.2 ± 0.4 min for the conjugate and 46.0 ± 0.6 min for the free DNA. The yield of the conjugate is 51 ± 6%. (B) The DNA duplex is 59-PEG4 -SPDP. The retention times are 28.3 ± 0.4 min for the conjugate and 45.0 ± 0.3 min for the free DNA. The yield of the conjugate is 94 ± 1%. (C) The DNA duplex is 59-PEG12 -SPDP. The retention times are 28.6 ± 0.4 min for the conjugate and 44.9 ± 0.2 min for the free DNA. The yield of the conjugate is 95 ± 2%.

chromatography of the crosslinking mixture using the same eluent without glycerol. The components of the reaction mixture were separated with similar efficiency and resolution in both cases (data not shown). Thus, we conclude that 10% glycerol in the eluent is tolerable for our application. The chromatogram with detection at 260 nm was used to measure retention time and area of each peak. The experiments with each type of modified DNA were repeated three times. The retention times of the peaks containing the MutS–DNA conjugate were found almost equal (differ less than 2%), not only among the conjugates containing similarly modified DNA but among all the conjugates studied. Neither shortening the linker (in the case of the SPDP-modified DNA), nor using fluorescently labeled DNA had a significant impact onto the conjugate retention time. Thus, the developed protocol proved to give very reproducible results which can be explained by the nature of the anion exchange chromatography. Since anion exchange separation of DNA and its conjugate with protein is based on the negative charges of the DNA phosphate groups, the presence or absence of fluorophores or other modifications coupled to the heterocyclic bases should not change the DNA affinity to the chromatographic resin. However, the retention time does depend on the DNA length. Therefore, an attempt to repeat such conjugate purification using DNA of another length

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25

Fig. 5. Separation of reaction mixture of MutS-497 conjugate with double-labeled 59-PEG4 -SPDP DNA duplex by ion exchange chromatography with detection at four different wavelengths (260 and 280 nm in panel A, 490 and 590 nm in panel B). The retention times are 28.3 ± 0.4 min for the conjugate and 45.0 ± 0.3 min for the free DNA. The yield of the conjugate is 94 ± 1%. (C) Analysis of the eluted fractions by 6% SDS–PAGE followed by Coomassie blue staining. Only one MutS subunit forms covalent bond with the reactive DNA. The MutS dimer dissociates under the conditions of SDS–PAGE. Therefore, the crosslinked complex yields two bands, the free MutS subunit and the other MutS subunit crosslinked to DNA.

would require some modifications in the protocol, namely changing the steps of the salt gradient (a conjugate with a longer DNA would elute at a higher salt concentration). Thus, we consider the developed protocol universal for purifying conjugates of different MutS variants with DNA duplexes of the same length carrying various modifications. The general scheme of the protocol could be useful as well for obtaining crosslinked complexes with different proteins. Yet, a disadvantage of this protocol is using high salt concentrations. As we show below, MutS retains its activity under such conditions. However, an attempt to use high salt concentration for another protein could result in its activity loss. The ratio of the two peak areas in the chromatogram corresponded to the ratio of the crosslinked and non-crosslinked DNA. The yield of the crosslinked complex was 51 ± 6% in the case of the SPDP-modified DNA, while for the other duplexes it was over 90% (Fig. 4). Thus, the crosslinking efficiency was proved to depend on the linker length, in agreement with the results obtained by SDS–PAGE (Fig. 3). To estimate the concentration of the unlabeled DNA–protein conjugate, an absorption spectrum was recorded. It was compared with the spectrum of the initial DNA duplex in a fixed concentration. This comparison did not give a completely precise concentration of the conjugate since the protein component of the complex introduced some additional absorbance at 260 nm. For the fluorescently labeled crosslinked complexes, we measured fluorescence of the acceptor fluorophore, Alexa-594, after its direct excitation at 590 nm. It was compared with the same signal obtained from the initial double-labeled DNA duplex in a fixed concentration. The protein did not emit light around 613 nm and therefore we considered the obtained concentration of the crosslinked complex to be precise. 3.6. Checking the functional activity of MutS in its crosslinked complex with DNA Since MutS is known to bind and bend mismatch-containing DNA molecules in the absence of ATP [10–12], we expected DNA

to be bent in the conjugate. ATP addition should result in conformational changes which lead to DNA unbending and sliding clamp formation [12,34,45]. We used FRET to monitor DNA unbending in the crosslinked complex. When the donor fluorophore is excited, some of its energy is transmitted to the acceptor fluorophore and the acceptor starts emitting light. If DNA gets bent, the distance between the fluorophores decreases and the acceptor fluorescence rises. This technique was recently applied for studying DNA bending by MutS [18,37,40]. We expected to see the opposite process during the DNA unbending, i.e., falling of the acceptor fluorescence. The fact of DNA unbending in response to ATP addition was considered an evidence of the fact that MutS had retained its activity after the crosslinking, complex purification and complex storage under conditions of high ionic strength. At first, FRET was measured under steady-state conditions. Donor (Atto-488) excitation in the crosslinked complex resulted in two emission peaks (Fig. 6A), at 519 nm (the donor itself) and at 613 nm (the acceptor Alexa-594). The ratio of the peaks was 0.37 (Fig. 6B). Then ATP was added which resulted in the increase of the first peak and decrease of the second peak; the peak ratio became 0.28. This change indicated increased distance between the two fluorophores testifying the DNA unbending. Thus, MutS proved to be active in the crosslinked complex. Then DTT was added in order to disrupt the disulfide bond which kept together the components of the crosslinked complex and to release MutS. It further decreased the peak ratio (up to 0.23). However, some MutS molecules could remain bound to the mismatch due to non-covalent interactions. Finally, proteinase K was added in order to destroy the protein completely. It decreased the peak ratio up to 0.16, testifying that MutS, once bound to a mismatch, does not dissociate rapidly. Additionally, we registered anisotropy of Atto-488 fluorescence during this experiment. Atto-488 is coupled to DNA through a linker and therefore is not expected to have a high anisotropy in a free DNA. Upon the complex formation with MutS, Atto-488 anisotropy increases since the protein restrains the fluorophore mobility to a certain degree. Destroying the crosslinked complex with DTT and proteinase K should decrease the anisotropy again. Indeed,

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Fig. 6. Checking activity of the crosslinked complex after its purification by ion exchange chromatography. (A) Fluorescence emission spectra. (B) Ratio of the acceptor fluorescence to the donor fluorescence at their maximums. (C) Anisotropy of Atto-488.

Atto-488 anisotropy was relatively high in the crosslinked complex (0.068), and then it decreased to 0.062 after ATP addition and to 0.057 after DTT addition (Fig. 6C). Thus, the ATP-induced conformational change in the crosslinked complex altered the relative positioning of MutS and Atto-488 providing more freedom for the fluorophore. Disruption of the disulfide bond by DTT could release some MutS molecules from DNA. Finally, destroying MutS with proteinase K resulted in the lowest anisotropy, 0.048. 3.7. ATP-dependence of the DNA unbending in the crosslinked complex After detecting the DNA unbending under steady-state conditions, we explored kinetics of this process. A careful study of

kinetics requires reducing amount of simultaneous processes as much as possible. Therefore, a crosslinked complex is the best object for such experiments. A similar MutS–DNA complex without a crosslink would demonstrate not only DNA unbending, but also MutS sliding along DNA, dissociation from DNA, reassociation and perhaps additional bending–unbending cycles. The stopped-flow technique allowed us to obtain kinetic curves over a broad range of ATP concentrations, from 1 ␮M to 1 mM (Fig. 7). Each curve was fitted with a single-exponential function (y = y0 + Ae−x/t ). The higher was ATP concentration, the faster was decrease in the acceptor fluorescence which corresponded to the DNA unbending: 0.023 ± 0.001, 0.17 ± 0.01, 2.1 ± 0.1, and 8.2 ± 0.2 s−1 for 1, 10, 100, and 1000 ␮M ATP (the ± values represent standard errors of the Origin fit). Similarly, the rate of sliding clamp formation and the rate of MutS release from a mismatch were shown to raise with increasing ATP concentration [46,23]. Taken together, these data suggest that ATP binding, rather than DNA unbending, could be rate-limiting for initiation of the subsequent MMR steps. 4. Conclusions

Fig. 7. Kinetics of DNA unbending in the crosslinked complex upon its mixing with different ATP concentrations.

The crosslinking approach is a popular technique for fixing macromolecular complexes in certain conformations. However, it is debatable if the protein components retain their natural activities in the resulting crosslinked complexes. In the present work, we used a simple reaction of thiol–disulfide exchange for producing MutS–DNA conjugate with a high yield. For the first time, anion exchange chromatography was utilized to separate this crosslinked complex from all the other components of the reaction mixture. A FRET-based assay was used to prove that the MutS protein in the purified conjugate retained its capability to bend and unbend DNA. The rate of DNA unbending in response to ATP addition was found to increase with increasing ATP concentration. The possibility to obtain and purify MutS conjugates with extended reactive

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DNA containing FRET pair would allow in the long term to investigate the combined action of MutS with the other proteins included in the MMR process. Acknowledgements We would like to thank Prof. Titia Sixma and her co-workers for kindly providing us with the MutS variant. This research was supported by Deutsche Forschungsgemeinschaft Grant GRK 1384 (to P.F. and A.H.), Russian Foundation for Basic Research Grant RFBRDFG 14-04-91343 (to M.M. and T.O.) and RFBR Grant 13-04-00615 (to A.R. and E.K.). References [1] A. Banerjee, W. Yang, M. Karplus, G.L. Verdine, Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA, Nature 434 (2005) 612–618. [2] C.G. Yang, C. Yi, E.M. Duguid, C.T. Sullivan, X. Jian, P.A. Rice, C. He, Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA, Nature 452 (2008) 961–965. [3] K.A. Malecka, W.C. Ho, R. Marmorstein, Crystal structure of a p53 core tetramer bound to DNA, Oncogene 28 (2009) 325–333. [4] C. Yi, G. Jia, G. Hou, Q. Dai, W. Zhang, G. Zheng, X. Jian, C.G. Yang, Q. Cui, C. He, Iron-catalyzed oxidation intermediates captured in a DNA repair dioxygenase, Nature 468 (2010) 330–333. [5] T.H. Tahirov, N.D. Babayeva, K. Varzavand, J.J. Cooper, S.C. Sedore, D.H. Price, Crystal structure of HIV-1 Tat complexed with human P-TEFb, Nature 465 (2010) 747–751. [6] N. Opalka, J. Brown, W.J. Lane, K.A. Twist, R. Landick, F.J. Asturias, S.A. Darst, Complete structural model of Escherichia coli RNA polymerase from a hybrid approach, PLoS Biol. 8 (2010) e1000483. [7] A.P. Zhang, Y.Z. Pigli, P.A. Rice, Structure of the LexA–DNA complex and implications for SOS box measurement, Nature 466 (2010) 883–886. [8] T. Ochi, B.L. Sibanda, Q. Wu, D.Y. Chirgadze, V.M. Bolanos-Garcia, T.L. Blundell, Structural biology of DNA repair: spatial organisation of the multicomponent complexes of nonhomologous end joining, J. Nucleic Acids 2010 (2010) 621695. [9] K. Fukui, DNA mismatch repair in eukaryotes and bacteria, J. Nucleic Acids 2010 (2010), Article ID: 260512. [10] M.H. Lamers, A. Perrakis, J.H. Enzlin, H.H. Winterwerp, N. de Wind, T.K. Sixma, The crystal structure of DNA mismatch repair protein MutS binding to a G × T mismatch, Nature 407 (2000) 711–717. [11] G. Obmolova, C. Ban, P. Hsieh, W. Yang, Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA, Nature 407 (2000) 703–710. [12] G.L. Hura, C.L. Tsai, S.A. Claridge, M.L. Mendillo, J.M. Smith, G.J. Williams, A.J. Mastroianni, A.P. Alivisatos, C.D. Putnam, R.D. Kolodner, J.A. Tainer, DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals, Proc. Natl. Acad. Sci. U. S. A. 11 (2013) 17308–17313. [13] C. Ban, M. Junop, W. Yang, Transformation of MutL by ATP binding and hydrolysis: a switch in DNA mismatch repair, Cell 97 (1999) 85–97. [14] A. Guarné, S. Ramon-Maiques, E.M. Wolff, R. Ghirlando, X. Hu, J.H. Miller, W. Yang, Structure of the MutL C-terminal domain: a model of intact MutL and its roles in mismatch repair, J. EMBO 23 (2004) 4134–4145. [15] T. Yamamoto, H. Iino, K. Kim, S. Kuramitsu, K. Fukui, Evidence for ATPdependent structural rearrangement of nuclease catalytic site in DNA mismatch repair endonuclease MutL, J. Biol. Chem. 286 (2011) 42337–42348. [16] L. Giron-Monzon, L. Manelyte, R. Ahrends, D. Kirsch, B. Spengler, P. Friedhoff, Mapping protein–protein interactions between MutL and MutH by crosslinking, J. Biol. Chem. 279 (2004) 49338–49345. [17] R. Ahrends, J. Kosinski, D. Kirsch, L. Manelyte, L. Giron-Monzon, L. Hummerich, O. Schulz, B. Spengler, P. Friedhoff, Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry, Nucleic Acids Res. 34 (2006) 3169–3180. [18] I. Winkler, A.D. Marx, D. Lariviere, R.J. Heinze, M. Cristovao, A. Reumer, U. Curth, T.K. Sixma, P. Friedhoff, Chemical trapping of the dynamic MutS–MutL complex formed in DNA mismatch repair in Escherichia coli, J. Biol. Chem. 286 (2011) 17326–17337. [19] M. Cristovao, E. Sisamakis, M.M. Hingorani, A.D. Marx, C.P. Jung, P.J. Rothwell, C.A. Seidel, P. Friedhoff, Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA, Nucleic Acids Res. 40 (2012) 5448–5464. [20] C. Jeong, W.K. Cho, K.M. Song, C. Cook, T.Y. Yoon, C. Ban, R. Fishel, J.B. Lee, MutS switches between two fundamentally distinct clamps during mismatch repair, Nat. Struct. Mol. Biol. 18 (2011) 379–385.

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