Study of the Lyase Activity of Human DNA Polymerase Î² Using

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Study of the Lyase Activity of Human DNA Polymerase # Using Analogues of the Intermediate Schiff Base Complex Sasha M Daskalova, Xiaoguang Bai, and Sidney M. Hecht Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00308 • Publication Date (Web): 06 Apr 2018 Downloaded from http://pubs.acs.org on April 8, 2018

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Biochemistry

Study of the Lyase Activity of Human DNA Polymerase β Using Analogues of the Intermediate Schiff Base Complex Sasha M. Daskalova, Xiaoguang Bai, and Sidney M. Hecht* Biodesign Center for BioEnergetics and School of Molecular Sciences, Arizona State University, Tempe, Arizona 85287, United States

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ABSTRACT: DNA polymerase β (Pol β) participates in mammalian base excision repair (BER). The enzyme has a two-domain architecture, reflecting its dual functionality. The polymerase activity, which replaces damaged nucleosides removed during an initial excision process, is within the C-terminal 31 kDa domain, while the N-terminal 8 kDa domain participates in a lyase function, working to remove a 5′-deoxyribose phosphate (5′-dRP) moiety from the damaged DNA substrate. The currently accepted mechanism of the lyase reaction involves a transient covalent enzyme‒DNA intermediate in the form of a Schiff base connecting Lys72 of the enzyme with the 5′-dRP moiety. The Schiff base intermediate is resolved via a β-elimination reaction, initiated by abstraction of a C2′-H atom from the 5′-dRP moiety. Presently, we describe the preparation of three Pol β enzymes modified at position 72 with aminoxy or hydrazinyl analogues of lysine. These enzymes form transient covalent bonds with the 5′-dRP moiety of the damaged DNA, in the form of an oxime and hydrazine, respectively. Both types of enzyme DNA intermediates are ultimately resolved by the lyase activities of each of the modified enzymes. Unsurprisingly, the formation and resolution of these E‒S complexes proceed with diminished kinetics, and with an altered pH profile. The experiments carried out provide additional support for Schiff base formation as an obligatory intermediate on the pathway to DNA repair, as well as for the proposed participation of Lys72 in effecting opening of the 5′-dRP ring via protonation of the ring oxygen atom, and for complex resolution via a β-elimination reaction. 2 ACS Paragon Plus Environment

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Biochemistry


Biochemistry

Human DNA polymerase β (Pol β) is a single-chain protein (~39 kDa ) that folds into two structurally and functionally discrete domains (Figure 1A)1 and has a key role in the singlenucleotide base excision repair (BER) pathway.2 The N-terminal 8 kDa domain of the enzyme participates in removal of the 5′-deoxyribose phosphate (5′-dRP) moiety generated after excision of the damaged base by DNA glycosylase and incision of the abasic site by apurinic/apyrimidinic (AP) endonuclease 1 (APE1). The C-terminal 31 kDa domain continues DNA repair by adding the correct complementary nucleotide to fill the generated gap.3‒5 The final step in restoration of the native structure of the nicked DNA is mediated by DNA ligase (Figure 1B).

A

P

A T

U G

C G

A T

G C

A T

URACIL-DNA GLYCOSYLASE

A T

G

C G

A T

G C

APE1 ENDONUCLEASE

A T

G

C G

A T

C G

G

A T

G C

DNA-POLYMERASE β

DNA-POLYMERASE β

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G C

OH P

A T

C G

C G

A T

G C

A T

G C

LIGASE 3

A T

C G

C G

Figure 1. Structure and enzymatic activities of human DNA polymerase β (Pol β). (A) Structure of human Pol β complexed with a double-stranded DNA having a 1-nucleotide gap (PDB 1bpy1). The 8 kDa lyase domain is colored in pink, the polymerase domain in green, and the DNA substrate is colored in beige. The key nucleophile in the active site of the lyase domain, Lys72, is colored in dark blue; (B) Schematic diagram of the base excision repair (BER) process. The lyase reaction is the rate-limiting step of the repair6 as kcat of the dRP lyase activity is approximately 10-fold lower than kcat for DNA synthesis7 and 100-fold lower than kcat of AP endonuclease activity.8 It is thought that the lyase reaction proceeds via β-elimination.4,9 The lyase catalytic pocket of Pol β, bracketed by the side chains of Glu26, Ser30, Lys35, Tyr39, Lys68, Lys84 and the primary nucleophile Lys72, is believed to retain its relatively rigid conformation after the initial binding of the abasic DNA substrate.10 Subsequent positional readjustment of the sugar associated with the damaged base to bring C1′ to a catalytically 4 ACS Paragon Plus Environment

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Biochemistry

relevant distance from Lys72 relies entirely on its own flexibility10,11 and does not perturb the existing interactions among the residues important for catalysis. Nε of Lys72 has been suggested to participate directly in the protonation of O4′ which induces sugar ring opening and enables a nucleophilic attack at C1′, resulting in formation of an imino (Schiff base) intermediate.11 Hydrogen bonding of Lys72 to the neighboring Tyr39 residue maintains the stability of the neutral Nε. The imine at C1′ lowers the pKa of the C2′-H, thus facilitating water-mediated abstraction of proton at C2′ with participation of Ser30-hydrogen bonded Glu26. This triggers scission of the C3′−O phosphodiester bond and removal of 5′-dRP group from the Schiff base complex. Previously, we studied a series of lysine analogues to explore the effect of slight alterations in nucleophilicity and position of the ε‐amino group of the primary catalytic residue Lys72 on the outcome of the lyase reaction.12 We found that the introduction of an Nε-methyl substituent had very little effect on the lyase activity of the modified Pol β, while alteration of side chain length or the nature of the nucleophile in the side chain of the residue at position 72 caused more substantial changes in the lyase function of the resulting enzyme. In the present study, three new lysine analogues were synthesized and incorporated into Pol β as probes of the mechanism of the lyase reaction. Characterization of the modified enzymes indicated that each formed an E−S complex with a DNA substrate containing a 5′-dRP moiety, and that each of the formed covalent binary complexes was resolved by the lyase activity of the enzyme. Interestingly, the modified amino acids in the three complexes differed significantly from lysine in the pKa of the nucleophilic atom putatively responsible for 5′-dRP ring opening/covalent bond formation, as well as in the stability of the covalent linkage whose formation was anticipated in analogy with Schiff base formation, and the effect of this linkage on


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the facility of abstraction of C2′-H. The functional nature of the novel lyase E−S complexes provides an important opportunity to dissect the dependence of lyase function on the AP site characteristics. EXPERIMENTAL PROCEDURES Chemical Synthesis of the Fully Protected pdCpA Derivatives of Amino Acids 1, 2 and 3. The chemical syntheses of (protected) amino acids 1 – 3 and their pdCpA derivatives are outlined in Schemes 1, S1, S2 and S3. Biochemical Experiments. DNA Template. The coding sequence of H. sapiens Pol β (NCBI, Accession No NM_002690) was E. coli codon-optimized (the codon adaptation index increased from 0.63 to 0.89), and cloned as a stop codon-less NcoI/XhoI insert into pET28b expression vector (Novagen), resulting in the placement of a hexahistidine tag at the protein Cterminus. Small-scale plasmid DNA isolation was performed using a GenElute Plasmid Miniprep Kit (Sigma). The Lys72 codon (AAG) was converted to an amber stop codon (TAG) using QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) using primer 5′GGCACCAAAATCGCAGAATAGATCGATGAATTTCTGGCTACGGG-3′ and its complementary strand. Maxi-preparations of mutagenized and wild-type pET28b:Pol β vectors for in vitro protein synthesis were done using an E.Z.N.A. Plasmid DNA Maxi Kit (Omega Biotek). DNA samples were additionally subjected to extraction with 25:24:1 phenol‒chloroform‒ isoamyl achohol, buffered with Tris to pH 8.0, and then ethanol precipitated, and were then resuspended in RNase-free water to a final concentration of 1 mg/mL. Preparation of E. coli S-30 Extract. A single colony of BL21(DE3) E. coli was freshly grown in 3 mL of LB medium for 3 h at 37 oC and 190 rpm; these cells were used to inoculate 200 mL of LB supplemented with 0.5 mM IPTG. The culture was then incubated at 37 oC and 190 rpm


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until an OD600 of 0.8‒0.9 was reached. The cells were collected by centrifugation at 3500g for 15 min at 4 oC, then weighed, washed three times with 20 mL/g of S30A buffer [14 mM MgOAc, 60 mM KOAc, 10 mM TrisOAc (pH 8.2), containing 0.01M DTT and 500 µL/L βmercaptoethanol], and once with 10 mL/g of S30B buffer (same as S30A but with 50 µL/L of βmercaptoethanol), and then resuspended in 1.27 mL/g of S30C buffer (S30A buffer without βmercaptoethanol). Each 1 mL of bacterial suspension was mixed with 0.3 mL of preincubation mix [0.32 M TrisOAc, pH 8.2, 9.38 mM MgOAc, 13.4 mM ATP, 14.7 mM GTP, 84 mM potassium phosphoenolpyruvate, 4 mM DTT, 48 µM amino acid mix], 1 µL of pyruvate kinase (15 U/µL), 2 µL of 1 M MgOAc and 2 µL of lysozyme (50 mg/mL). Samples were incubated at 37 oC for 40 min, then at ‒80 oC overnight, thawed at 37 oC for 40 min, transferred again at ‒80 o

C for 1 h and thawed at room temperature for 40 min. EGTA was added to a final concentration

of 2.5 mM followed by incubation at 37 oC for 30 min. CaCl2 was then added to a final concentration of 2.5 mM, and the samples were incubated at ‒80 oC for 1 h. Frozen cell lysates were centrifuged at 15,000g and 4 oC for 1 h. Supernatants were carefully transferred to fresh tubes and stored in small aliquots at ‒80 oC until use. Transcription and Purification of tRNA-COH, Followed by Ligation to PentenoylProtected Aminoacyl-tRNA. An abbreviated yeast phenylalanine tRNACUA-COH transcript was produced using an Ampliscribe T7 Transcription Kit (Epicentre Biotechnologies) from FokIdigested pYRNA8 DNA. Purification was effected by DEAE Sepharose CL-6B chromatography; elution with a 0.1 to 0.7 M step gradient of NaCl in 100 mM NaOAc, pH 5.0. The collected fractions were precipitated using isopropanol; the pellets were redissolved in RNase-free water and then analyzed by electrophoresis on 8% acidic (100 mM NaOAc, pH 5.0) polyacrylamide gels containing 7 M urea after staining with methylene blue. Ligation to the corresponding


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aminoacyl-pdCpA was performed at 37 oC for 1.5 h using 1 U/mL of T4 RNA ligase 1 (New England Biolabs), 20 µL of ligation buffer [50 mM Hepes buffer, pH 7.5, with 15 mM MgCl2], 1 µg/µL of tRNACUA-COH, 0.005 OD260/µL of aminoacyl-pdCpA and 15% (v/v) DMSO. Samples were ethanol precipitated, resuspended in water and deprotected by treatment with 12.5 mM iodine for 15 min at room temperature. Following a second ethanol precipitation, the pellets were redissolved in RNase-free water to a final concentration of 3 µg/µL. The efficiency of ligation was evaluated by acidic PAGE, as described above. Coupled in Vitro Transcription/Translation and Protein Purification. An analytical scale (10 µL) reaction mixture was assembled by mixing 4 µL of 2.5× premix [87 mM Tris, 476 mM potassium glutamate, 75 mM ammonium acetate, 20 mM MgOAc•4H2O, 63 mM potassium phosphoenol pyruvate, 2 mM IPTG, 8.64% (w/v) PEG 8000, 0.2 mg/mL of folinic acid, 2.5 mM cAMP, 1.25 mM UTP, 1.25 mM CTP, 5 mM ATP, 5 mM GTP, 5 mM DTT, and 0.48 mg/mL of E. coli tRNA, pH 7.4], 0.1 µL of RNase inhibitor (ThermoFisher Scientific; 40 U/µL), 0.1 µL of 100× protease inhibitor cocktail (Roche), 0.2 µL of 100 mg/mL rifampicin, 2.6 µL of E. coli S30 extract, 0.5 µL of 0.3 mM amino acid mix without methionine, 0.5 µL of [35S]Met (10.2 mCi/mL), 0.5 µL (0.5 mg) of plasmid DNA and 1.5 µL (3 mg/mL) of aminoacylated suppressor tRNACUA. For enzyme purification, the volume was scaled up as required by the suppression yield. Controls with tRNACUA-COH were run in parallel. All reactions were incubated for 1 h at 37 oC, mixed with an equal volume of 2× SDS loading buffer [62.5 mM Tris-HCl, pH 6.8, containing 25% glycerol, 2% SDS, 0.72 M β-mercaptoethanol and 0.1% Orange G], heated for 5 min at 95 oC and analyzed by SDS−PAGE on 15% acrylamide gels. The gels were fixed for 1 h with 40:10:50 ethanol‒acetic acid‒water, and then for another hour with 20:10:70 ethanol‒acetic acid‒water, and exposed overnight. Autoradiograms were scanned on a Storm Scanner 820


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Biochemistry

(Amersham Biosciences) phosphorimager and quantified with Image Quant software Version 5.2. In vitro synthesized proteins were purified by immobilized metal affinity chromatography (IMAC) on Talon resin. The in vitro reaction was subjected to buffer exchange with 40 mM Hepes buffer, pH 7.5, containing 300 mM NaCl and 7.5 mM imidazole by the use of a Pierce desalting column (ThermoFisher Scientific). The sample was mixed with Talon resin (Clontech) that had been equilibrated with the same buffer in 5:1 (v/v) ratio and then rotated at 4 oC for 1 h. The suspension was poured into a column and washed with 20 column volumes of 40 mM Hepes, pH 7.5, containing 300 mM NaCl and 7.5 mM imidazole. The bound proteins were eluted with three column volumes of 40 mM Hepes buffer, pH 7.5, containing 150 mM NaCl and 250 mM imidazole, and then concentrated (10 K Amicon Ultra Centrifugal filters, Millipore) and subjected to buffer exchange to 100 mM Hepes buffer, pH 7.5, containing 50 mM KCl, 0.2 mM EDTA, 2 mM DTT and 0.01 mg/mL of BSA. An equal volume of glycerol was then added and the sample was stored at ‒20 oC. Preparation of 5′-dRP−Labeled Substrate. The 34-mer oligodeoxyribonucleotide containing uridine at position 16 (5′-CTGCAGCTGATGCGCUGTACGGATGCCCGGGTAC3′) was 3′-end labeled using terminal transferase (Roche Life Science) using 3′-[α-32P] cordycepin 5′-triphosphate (Perkin Elmer) according to the manufacturer’s protocol. Non-bound radioactive nucleoside was removed with an Illustra MicroSpin G25 column (GE Healthcare). Labeled oligonucleotide was annealed to its complementary strand (5′GTACCCGGGCATCCGTACGGCGCATCAGCTGCAG-3′) in 10 mM Hepes buffer, pH 7.5, containing 50 mM KCl and 5 mM MgCl2, by heating at 94 oC for 3 min then cooling slowly to 25 oC. To prepare the substrate for the enzyme assays by removing the uracil base and cleaving


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the phosphodiester backbone immediately 5′ to the AP site, the DNA duplex was treated with 1 U per pmol DNA of both uracil-DNA glycosylase (UDG, NEB) and APE1 (NEB) for 10 min at 37 oC in 10 mM Hepes buffer, pH 7.5, containing 50 mM KCl, 5 mM MgCl2, and 1 mM DTT. Due to the labile nature of the AP site, the prepared 5′-dRP DNA substrate was used immediately in the assays. Lyase and BER Assays. The lyase reaction was typically performed at room temperature in 30 µL of 30 mM Hepes buffer, pH 7.5, containing 25 nM enzyme and 40 nM substrate, as well as 25 mM KCl, 5 mM MgCl2, and 1 mM DTT. Aliquots (9 µL) were taken at 1, 15 and 30 min and mixed with 1 µL of aqueous NaBH4 to a final concentration of 100 mM NaBH4. After incubation on ice for 30 min, an equal volume of formamide dye (95% formamide, 20 mM EDTA, pH 8.0, containing 0.025% bromophenol blue) was added and samples were heated for 3 min at 95 oC. Two-µL aliquots were loaded on a denaturing 20% sequencing polyacrylamide gel containing 7 M urea (40 × 32 × 0.04 cm) and run for 7-7.5 h at 2000 V (constant voltage). Visualization was done by autoradiography. The BER assay was typically performed by incubating 50 nM 5′-dRP DNA substrate and 15 nM enzyme with T4 DNA ligase (NEB) in 15 µL (total volume) of 50 mM Hepes buffer, pH 7.8, containing 75 mM KCl, 10 mM MgCl2, 2 mM DTT, 0.5 mM EDTA, 2 mM ATP, and 0.02 mM dATP, dCTP, dGTP and dTTP. The reaction mixture was incubated for 10 min at 37 oC. An equal volume of formamide dye (95% formamide, 20 mM EDTA, pH 8.0, containing 0.025% bromophenol blue) was then added and the samples were heated at 70 oC for 2 min before performing PAGE on a 20% denaturing 7 M urea gel, followed by autoradiography for visualization.


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Trapping of E‒S Intermediates. Trapping of the DNA−enzyme complex that is an intermediate of the lyase reaction was performed on ice in 30 mM Hepes buffer, pH 7.5, supplemented with 50 mM KCl, 5 mM MgCl2 and 1 mM DTT. Ice-cold 5′-dRP DNA substrate (100 nM) and enzyme (25 nM) were mixed in the presence of 20 mM NaBH4. The reaction was terminated at different time points by mixing an aliquot with an equal volume of 2× SDS loading buffer. In a parallel experiment, the lyase reaction was allowed to proceed for 1, 10 or 30 min on ice before addition of NaBH4 to a final concentration of 20 mM. This was followed by incubation on ice for another 30 min to stabilize the product via imine reduction and mixing with an equal volume of 2× SDS loading buffer. In each case, the samples were analyzed by SDS−PAGE on 15% polyacrylamide gels, then fixed and processed as described above. In order to enable a study of the effect of pH on E‒S intermediate formation and resolution, the original Hepes buffer, pH 7.5, was replaced with a series of buffers (sodium acetate buffer, pH 4.5 and 5.5, sodium phosphate buffer pH 6.5, Hepes buffer pH 8.5, sodium carbonate/bicarbonate buffer pH 9.5 and 10.5) and the experiments were carried out as described above. RESULTS Preparation of pdCpA and tRNACUAs Activated with Lysine and Three Lysine Analogues. The preparation of suppressor tRNACUA transcripts activated with lysine analogues 1, 2 and 3 began with S-glutamic acid. Functional group transformations enabled conversion to fully protected amino acids which were activated as their respective cyanomethyl esters and coupled to pdCpA as esters 14, 23 and 29, respectively. The synthetic route employed for 14 (the pdCpA ester of fully protected 1) is outlined in Scheme 1. As shown, S-glutamic acid was converted to its bis-methyl ester (4) by treatment with thionyl chloride in anhydrous methanol in quantitative yield. Treatment with (Boc)2O then afforded N-


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Scheme 1. Synthesis of the Fully Protected pdCpA Derivative of δ-Aminooxynorvaline (1)

Boc-S-glutamic acid dimethyl ester 5 in 86% yield.13 In order to achieve selective reduction of the γ-ester, a second N-Boc group was introduced (DMAP, Boc2O, 100% yield) to provide 6, and the relatively bulky reducing agent diisobutylaluminium hydride (DIBAL-H) was employed. The desired alcohol 7 was obtained in 67% yield. Also obtained in 25% yield during the conversion 6 → 7 was the respective aldehyde (16, Scheme S1), which proved to be a key intermediate for the syntheses of the pdCpA esters of 2 (23, Scheme S2) and 3 (29, Scheme S3). Completion of the synthesis of pdCpA ester 14 was accomplished by conversion of alcohol 7 to the corresponding bromide 8 (CBr4, PPh3, 62% yield), followed by treatment with N-Bochydroxylamine, which provided 9 as a colorless oil in 41% yield. The N-Boc protecting groups were removed (CF3COOH, CH2Cl2) and 10 was protected with two N-pentenoyl protecting 12 ACS Paragon Plus Environment

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Biochemistry

groups to give 11 in 66% overall yield for two steps. Demethylation of the ester moiety permitted conversion to cyanomethyl ester 13 in an overall yield of 44%. Finally, the cyanomethyl ester was coupled with pdCpA tetrabutylammonium salt to afford the pdCpA ester 14, albeit only in low yield. Ester 14 was incubated with a suppressor tRNACUA-COH transcript in the presence of T4 RNA ligase + ATP, affording the bis-N-protected δ-aminooxynorvalyltRNACUA (15), in analogy with earlier studies.12 The preparation of δ-methylhydrazinylnorvalyltRNACUA (24) (Scheme S2) and δ-hydrazinylnorvalyl-tRNACUA (30) (Scheme S3) were carried out using a similar strategy. Both employed key intermediate 16 for introduction of the hydrazine functionality. The outcome of the ligation reactions was monitored by acidic gel electrophoresis14 and methylene blue staining. As shown in Figure 2B, lysyl-tRNACUA was cleanly resolved from unreacted suppressor tRNACUA-COH transcript. In comparison, the separation of the tRNAs activated with lysine analogues 1 – 3 from tRNACUA-COH was less definitive, especially for the putative δ-methylhydrazinylnorvalyl-tRNACUA. While this failed to provide confirmation that the ligation reaction had been successful, the subsequent protein synthesis experiments carried out

Figure 2. In vitro synthesis of modified Pol β enzymes. (A) Structures of three lysine analogues incorporated into position 72 of human Pol β; (B) Ligation of modified lysyl-pdCpA derivatives to an abbreviated suppressor tRNACUA-COH transcript (Scheme 1) monitored by acidic PAGE and methylene blue staining; (C) Analytical scale in vitro synthesis of human Pol β modified at position 72, as monitored by SDS−PAGE and autoradiography. Suppression yields were calculated based on band intensity readings. 13 ACS Paragon Plus Environment

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with these activated suppressor tRNAs were entirely successful, indicating that the ligation reactions actually had taken place. Synthesis of Modified Human DNA Polymerases β Containing Lysine Analogues 1 – 3 at Position 72. Initially, analytical scale in vitro synthesis of the corresponding modified Pol β enzymes was performed to estimate the suppression levels. Relative to lysyl-tRNACUA , whose level of suppression of the UAG codon at position 72 was defined as 100%, suppressor tRNACUAs activated with analogues 1 – 3 afforded suppression yields of 69, 94 and 78%, respectively. Repetition of this experiment gave comparable suppression values for the three lysine analogues (not shown). Critically, in the presence of the abbreviated suppressor tRNACUACOH, lacking any amino acid at the 3′-end, only the truncated form of the enzyme (MW 7.9 kDa) migrating below the 10 kDa marker was detected (Figure 2C, left lane labeled C). These observations, as well as confirmation by MS/MS in our previous studies,12,15 indicated that the in vitro synthesis system is robust and affords negligible levels of amino acid misincorporation at the UAG codon. Wild type and modified enzymes were further studied in coupled in vitro transcription/translation reactions. The elaborated Pol β enzymes contained fusion peptides at the C-terminus of the enzyme which included a hexahistidine sequence. This enabled purification by IMAC on Talon resin, after which the isolated proteins were analyzed by SDS-PAGE and Imperial Coomassie staining. As shown in Figure S1 (left panel, second lane), the elaborated Pol

β co-eluted with a few endogenous E. coli proteins. When the 6xHis-tag was replaced with a Strep-tag and the in vitro synthesis was guided from the Strep-tagged template, the preparation was of slightly better quality (Figure S1, left panel, third lane). However, as the lyase activity was consistent regardless of the template used (not shown), all of the Pol β constructs were


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Biochemistry

produced using the more cost-effective 6xHis-tagged template and subsequently purified by Talon chromatography. Purified enzymes were quantitatively equalized based on incorporated 35

S-methionine (Figure S1, right panel).

Trapping of Reversibly Formed E−S Covalent Binary Complex Using Sodium Borohydride. The ability of the modified Pol β enzymes to form a covalent E−S intermediate with a damaged DNA known to be a substrate for wild-type Pol β was assessed by mixing each of the modified enzymes with the substrate on ice in the presence of 20 mM sodium borohydride. Borohydride can stabilize the E−S complex by reduction of the formed Schiff base and thereby prevent the lyase reaction from proceeding to completion, i.e. by blocking removal of the 5′-dRP group (Figure 1B). Therefore, the accumulation of the covalent E−S intermediate reflected both

Figure 3. Time course of trapping of E−S intermediate in the (constant) presence of sodium borohydride. Samples were analyzed by SDS−PAGE and autoradiography. (A) Wild-type Pol β; (B) Pol β containing δ-methylhydrazinylnorvaline (2) at position 72; (C) Pol β containing δaminooxynorvaline (1) at position 72; (D) Pol β containing δ-hydrazinylnorvaline (3) at position 72. The graph underneath each trace illustrates the time course of (covalent) E−S substrate accumulation throughout the monitored period (10 – 600 sec). The most intense band in each case was used as a reference (100%). 15 ACS Paragon Plus Environment

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the fitness of the catalytic nucleophile located at position 72 to form a Schiff base (analogue) with the DNA substrate, and also the facility of Schiff base (analogue) reduction by borohydride. Following admixture of Pol β with a damaged DNA duplex containing a dRP moiety (Figure 1B), aliquots were taken at several time points and analyzed by SDS−PAGE and autoradiography. While the wild-type Pol β was engaged in the complex almost immediately, reaching saturating levels within the first 10 seconds (Figure 3A), all three modified enzymes demonstrated compromised ability to form the intermediate and negligible amounts of E−S complex were detected at the onset of reaction (Figure 3B, C, D). The amount of covalent intermediate gradually increased for all three modified enzymes during the monitored time course at similar rates. To determine whether the individual enzymes could dissociate from the DNA substrate, the lyase reaction was allowed to proceed on ice for predetermined times before the addition of sodium borohydride. In this case, the intensity of the signal of the E−S intermediate at each time point should depend on several parameters: (i) the ability of the enzyme to form the E−S complex; (ii) the stability of the E−S complex; and (iii) the ability of the enzyme to remove the 5′-dRP group. As each reaction progresses, the signal for the covalent binary complex would be expected to diminish until disappearing completely once the reaction is complete. As expected for the wild-type enzyme, the binary complex band was detected only at the first 1 minute time point (Figure 4A, panel 1 from the left). In contrast, 30 min after initiation of the reaction, the modified enzymes were still complexed detectably with the labeled substrate (Figure 4A, panels 2-4 from the left). The decrease in signal intensity followed slightly different dynamics depending on the structure of the Lys72 analogue (Figure 4B), although the enzyme containing


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amino acid 2 at position 72 appeared to progress through resolution of the covalent binary complex more rapidly than the other two modified enzymes. Lyase and Base Excision Repair (BER) Activity of the Modified Enzymes. The ability of each of the modified Pol β enzymes to support BER was assessed in comparison to wild type. The BER reaction proceeded to completion, i.e. to restoration of the full length 34-nt DNA strand (Figure 5, lane 1) from the abasic, nicked substrate (Figure 5, lane 2) only in the presence of Pol β having functioning lyase and polymerase activities, and only in the additional presence of DNA ligase (Figure 5, cf lanes 3, 7 and 8). Wild-type enzyme performed BER very efficiently (Figure 5, lane 3). In contrast, the BER activity of the three modified enzymes was greatly reduced (Figure 5, lanes 4-6).

Figure 4. Trapping of the E−S intermediate by sodium borohydride reduction during the course of the lyase reaction. The lyase reaction was performed for the specified time (1, 10, or 30 min) before sodium borohydride addition. (A) Autoradiography traces; (B) Percentage of trapped intermediate trapped as (reduced) covalent complex. 17 ACS Paragon Plus Environment

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Figure 5. Base excision repair (BER) reaction with wild-type and modified Pol β enzymes monitored by PAGE using a 20% denaturing sequencing gel and autoradiography. The structures of the substrate (S) and the recovered product (P) are presented on the right and boxed. Reactions are numbered beneath the gel (lanes 1-8). Matsumoto et al.9 demonstrated that the Lys72Arg Pol β mutant retained the polymerase activity of wild type. Similarly, replacement of Lys72 with alanine did not affect DNA substrate binding nor the polymerase activity of the modified protein.16 Therefore, the overall BER efficiency of our modified Pol β enzymes was expected to reflect predominantly their ability to remove the 5′-dRP group. Indeed, the modified proteins demonstrated poor lyase activity (Figure 6A, B, C). The effect of replacing the catalytic lysine residue with analogues 1 – 3 was modeled by diluting the wild-type pol β and running the lyase experiment for a shorter period of time. To achieve a substrate:product ratio similar to that generated by the modified enzymes after 15 minutes, the wild-type enzyme had to be diluted 2 to 3-fold, and allowed to perform catalysis for only 1 minute (Figure 6D). The lyase activity of human Pol β has been shown to have a wide pH alkaline optimum.16 This was also true of the wild-type enzyme synthesized in vitro, which demonstrated good lyase 18 ACS Paragon Plus Environment

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Biochemistry

Figure 6. Lyase activity of modified Pol β enzymes monitored by PAGE using a 20% denaturing sequencing gel and autoradiography. (A) Time course for Pol β containing δ-aminooxynorvaline (1) at position 72; (B) Time course for Pol β containing δ-methylhydrazinylnorvaline (2) at position 72; (C) Time course for δ-hydrazinylnorvaline (3) at position 72. In each case, wild-type lyase activity was assessed in parallel with the corresponding control (substrate only). (D) Lyase activity of wild-type Pol β following serial dilutions (1-minute incubation). activity within the pH range 8.0 – 9.5 (Figure 7A). The pH dependence of the lyase reaction was also measured for the modified Pol β containing 2 at position 72; this enzyme displayed the

Figure 7. Effect of pH on lyase activity. Lyase activity pH optimum of (A) in vitro synthesized wild-type Pol β and (B) in vitro synthesized Pol β containing δ-methylhydrazinylnorvaline (2) at position 72, monitored by autoradiography following PAGE using a 20% denaturing sequencing gel.


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greatest lyase activity of the three modified enzymes (Figures 4 and 6). As shown in Figure 7B, Pol β containing δ-methylhydrazinylnorvaline (2) at position 72 also exhibited its greatest lyase activity within a similar alkaline pH range, perhaps not unexpectedly given the nature of the modification introduced. The effect of pH was studied further to encompass its influence on formation and resolution of the enzyme–DNA covalent binary complex. The formation of the E−S complex was monitored by autoradiography at two time points, 1 and 5 minutes, in the constant presence of sodium cyanoborohydride. In parallel with the increase of pH from 4.5 to 9.5, the wild-type enzyme demonstrated increased formation of the covalent binary complex. No significant difference between the intensity of the signals for the two monitored time points was detected at pH 7.5, 8.5

Figure 8. Effect of pH on the trapping of E-S intermediate. Trapping of the complex between wild-type DNA pol β or pol β containing δ-methylhydrazinylnorvaline (2) at position 72 and the DNA substrate was performed on ice in the constant presence of sodium cyanoborohydride for 1 or 5 min (upper panel). An aliquot of each sample was then mixed with an equal volume of 2× SDS sample buffer, analyzed on a 15% SDS polyacrylamide gel and visualized by autoradiography. Buffers used: pH 4.5 and 5.5, sodium acetate buffer; pH 6.5, sodium phosphate buffer; pH 7.5 and 8.5, Hepes buffer; pH 9.5 and 10.5, sodium carbonate/bicarbonate buffer. The 5-min data were used for the plot in the lower panel. 20 ACS Paragon Plus Environment

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Biochemistry

or 9.5, indicative of a broad optimum in this pH range (Figure 8, upper panel). A further increase in the pH to10.5 resulted in a drastic decline in the amount of the binary complex formed during the first minute of the reaction. Typical optimum binary complex levels were not completely achieved even after 5 minutes. Modified Pol β containing 2 at position 72 also favored alkaline conditions for formation of the E−S complex, but with a clearly defined the optimum at pH 7.5 (Figure 8, lower panel). However, it must be noted that for the entire pH range studied, including the pH optimum, the complex was formed only at negligible levels during the first minute, suggesting some mechanistic limitation to efficient complex formation. One further experiment measured in parallel the entire amount of the trapped E−S complex within the first 5 minutes and the amount that could be captured beginning 5 minutes after the onset of the lyase reaction. To this end, cyanoborohydride was added at the beginning of the experiment, as compared with its addition after 5 minutes. As shown in Figure 9 (upper panel), the wild-type enzyme demonstrated the greatest accumulation of the E−S complex in the constant presence of cyanoborohydride at alkaline pH. A 5-minute delay in the addition of cyanoborohydride afforded much lower, but still detectable signals for the E−S complex only at pH values of 7.5 and 10.5, i.e. at peripheral values within the optimal pH range for the lyase reaction (cf Figures 7 and 9). In comparison, modified pol β containing 2 at position 72, formed traces of the E−S complex in the constant presence of cyanoborohydride over the entire pH range tested, but very substantial amounts when the reducing agent was added 5 minutes after the beginning of the lyase reaction (Figure 9, lower panel). The increase occurred even at pH 9.5, the pH at which the greatest amount of binary complex was observed when cyanoborohydride was present from the beginning of the lyase reaction. This phenomenon of reversed intensity of the signals suggests that the modified enzyme forms the E−S complex much more slowly compared


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to the wild type, and at a rate much less than the rate of reduction of the AP sites in the ringopened aldehydic form by cyanoborohydride. Problems with 5′-dRP resolution by the modified enzyme could also have contributed to the observed outcome.

Figure 9. Effect of pH on E‒S intermediate formation and resolution. Trapping (T) of the complex between wild-type Pol β (gray squares) or pol β containing δmethylhydrazinylnorvaline (2) at position 72 (blue diamonds) and the DNA substrate was performed on ice for 5 min in the constant presence of sodium cyanoborohydride. In parallel, the reactions (R) were performed on ice for 5 min before the addition of sodium cyanoborohydride, followed by incubation on ice for an additional 30 min for complex stabilization. An aliquot of each sample was then mixed with an equal volume of 2× SDS sample buffer, analyzed on a 15% SDS polyacrylamide gel and visualized by autoradiography. Buffers used: pH 4.5 and 5.5, sodium acetate buffer; pH 6.5, sodium phosphate buffer; pH 7.5 and 8.5, Hepes buffer; pH 9.5 and 10.5, sodium carbonate/bicarbonate buffer.

DISCUSSION In a recent publication, we explored the effect of altering the side chain of Lys72 of human DNA polymerase β on the lyase and polymerase activities of the enzyme.12 The lyase activity is mediated through a reversibly formed covalent enzyme‒DNA intermediate, namely a Schiff base. Accordingly, most of the analogues in that study retained the amine functional group essential for Schiff base formation. The replacement of the primary side chain amine with a secondary amine (N-methyllysine) afforded an enzyme which most closely retained the characteristics of wild type. Side chain elongation was realized both by the introduction of homolysine and thialysine in lieu of Lys72. These two modified enzymes functioned reasonably well in the lyase assay, both at the levels of Schiff base formation, and the release of the 5′-dRP


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Biochemistry

moiety from the damaged DNA strand. In comparison, the imposition of conformational constraint by including the side chain amine within a piperidine ring (piperidinylalanine), or the replacement of the amine with a guanidine moiety (homoarginine), led to greatly diminished Schiff base formation, dRP excision and BER repair. In the present study, the focus has shifted to exploration of the need for a classical Schiff base intermediate. The key role of Lys72 as a primary nucleophile in the lyase reaction has been confirmed by a number of studies.9,16‒18 However, as we have previously demonstrated that Pol β has some tolerance for alteration of lysine side chain length and substitution,12 the functional tolerance of Pol β for alterations in the nature of the Schiff base seemed of interest for study. Logically, it might have been anticipated that the effect could depend on any of several parameters, including the position and nucleophilicity/basicity of the atom(s) replacing the lysine primary amine, as well as the ability of the modified enzyme to form a reversible covalent complex with its damaged DNA substrate, and to effect removal of the dRP moiety from the binary complex. Compared to lysine, δ-aminooxynorvaline (1) has an altered geometry. In free lysine, the Cα−Cβ, Cβ−Cγ, Cγ−Cδ and Cδ−Cε bond lengths are 1.524 Å, 1.518 Å, 1.526 Å and 1.521 Å, respectively, and the Cα−Cβ−Cγ, Cβ−Cγ−Cδ and Cγ−Cδ−Cε bond angles are 114.6o, 111.0° and 111.5o, respectively.19 In δ-aminooxynorvaline (1), the Cε atom is replaced by an oxygen atom. As the average C−O (1.43 Å) and N−O (1.36 Å) bond lengths are slightly shorter than C−C (1.53 Å) and C−N (1.43 Å) bond lengths, the Cα−Nε distance in 1 is also expected to be shorter compared to the in silico predicted 6.36 Å Cα−Nε distance for free lysine. The same Cα−Nε distance in 4-oxalysine was predicted in silico to be 6.09 Å and, considering their structural similarity, the corresponding value for δ-aminooxynorvaline (1) should be quite similar. A slight repositioning of the ε-amino group is also expected due to the difference


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between C−C−N and C−O−N bond angles. Additionally, the protonated aminooxy moiety has a lower pKa compared to the protonated primary amine. The pKa of the δ-aminooxynorvaline side chain is predicted to be 4.41 (ChemAxon, chemicalize.com); therefore, much lower compared to the pKa of 10.53 for the free lysine side chain amino group. Upon reaction with aldehydes and ketones, the formed oxime ether linkage is extremely robust, while the imine linkage is reversible and readily hydrolyzed in water.

Figure 10. Models of wild type Pol β (upper panel) and Pol β containing 2-amino-5hydroxypentanoic acid at position 72 (lower panel) after repositioning the 5′-dRP group of the DNA substrate to a catalytically relevant distance to Lys72 by a ̴ 120o rotation about the 3′phosphate.11 Nε-C1′, Nε-O4′, Cδ-C1′ and Cδ-O4′ distances are shown for the wild-type enzyme and Oδ-C1′ and Oδ-O4′ distances are shown for the modified enzyme. The original position of the 5′-dRP group relative to Lys72 is presented in the inset of the upper panel. Models of the wild-type (PDB entry 2P66) and modified Pol β enzymes were produced with Chimera 1.9 software.21 The SwissSideChain database20 was used to select the highest probability 2-amino-5hydroxypentanoic acid rotamer (P = 0.29). The ways in which the foregoing structural and electronic differences may be expected to impact the lyase and BER reactions seem worthy of consideration. Due to the shorter Cα−Nε


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Biochemistry

distance, the nucleophile of δ-aminooxynorvaline might be expected to reside slightly farther from the 5′-dRP moiety in the initially formed enzyme‒DNA non-covalent binary complex. This analysis is supported by in silico modeling results. Lysine analogue δ-aminooxynorvaline (1) is not available in the SwissSidechain database.20 However, structural comparison between 2amino-5-hydroxypentanoic acid-substituted and wild-type Pol β indicated that Oε−C1′ and Oε−O4′ distances in the modified enzyme were just slightly different than the corresponding Cε−C1′ and Cε−O4′ distances (Figure 10). Accordingly, it seems reasonable to expect that the Nε atom of the δ-aminooxynorvaline-substituted enzyme would also reside at a catalytically relevant distance from the 5′-dRP group. However, at physiological pH, the aminooxy group (predicted pKa 4.51) would not be protonated; hence, it should not be capable of facilitating sugar ring

Figure 11. (A) Putative mechanism for dRP excision mediated by the lyase domain of Pol β (adapted from ref 6). (B) Inferred structures of the covalent E−S intermediates formed from the modified Pol β enzymes containing amino acids 1, 2 and 3 at position 72.


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opening through protonation of O4′ 7,11, 22,23 (Figure 11A) if such is required. According to NMR studies, an apurinic (AP) site in DNA exists as an equilibrating mixture of three species: the αhemiacetal, β-hemiacetal and open chain aldehyde. The aldehyde is the most chemically reactive species, but represents only a small portion (

Study of the Lyase Activity of Human DNA Polymerase Î² Using

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