Probing the Flexibility of the Catalytic Nucleophile in the Lyase

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Probing the Flexibility of the Catalytic Nucleophile in the Lyase Catalytic Pocket of Human DNA Polymerase # with Unnatural Lysine Analogues Sasha M Daskalova, Chandrabali Bhattacharya, Larisa M Dedkova, and Sidney M. Hecht Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00807 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Probing the Flexibility of the Catalytic Nucleophile in the Lyase Catalytic Pocket of Human DNA Polymerase with Unnatural Lysine Analogues  Sasha M. Daskalova, Chandrabali Bhattacharya, Larisa M. Dedkova and Sidney M. Hecht*

Biodesign Center for BioEnergetics, and School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, United States

1

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 51

ABSTRACT: DNA polymerase β (Pol β) is a key enzyme in mammalian base excision repair (BER), contributing stepwise 5′-deoxyribose phosphate (dRP) lyase and 'gap-filling' DNA polymerase activities. The lyase reaction is believed to occur via a β-elimination reaction following the formation of a Schiff base between the dRP group at the pre-incised apurinic/apyrimidinic site and the ε-amino group of Lys72. To probe the steric constraints on the formation and subsequent resolution of the putative Schiff base intermediate within the lyase catalytic pocket, Lys72 was replaced with each of several nonproteinogenic lysine analogues. The modified Pol β enzymes were produced by coupled in vitro transcription/translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72. In the presence of a misacylated tRNACUA transcript, suppression of the UAG codon in the transcribed mRNA led to elaboration of full length Pol having a lysine analogue at position 72. Replacement of the primary nucleophilic amine with a secondary amine in the form of Nmethyllysine (4), affected mainly the stability of the Schiff base intermediate and resulted in relatively moderate inhibition of lyase activity and base excision repair. Elongation of the side chain of the catalytic residue by one methylene group, achieved by introduction of homolysine (6) at position 72, apparently shifted the amino group to a position less favorable for Schiff base formation. Interestingly, this effect was attenuated when the side chain was elongated by replacing one side chain methylene group with a bridging S atom (thialysine, 2). In comparison,

2


Page 3 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

replacement of lysine 72 with an analogue having a guanidine moiety in lieu of an -amino group (homoarginine, 5), or a sterically constrained secondary amine (piperidinylalanine, 3) led to almost complete suppression of dRP excision activity and the ability of Pol to support BER. These results help to define the tolerance of Pol  to subtle local structural and functional alterations.

3


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Base excision repair (BER) is a conserved mechanism for maintaining gene integrity and genomic stability of mammalian cells.1 Two major repair pathways, known as single-nucleotide BER and long-patch (2-10 nucleotides) BER, ensure removal of DNA lesions and modifications caused by endogenous and/or exogenous agents and factors.2 Regardless of differences in the enzymatic machinery, each of these pathways operates in five consecutive steps including recognition and removal of the damaged base, DNA incision at the abasic site, removal of the remaining sugar moiety, filling the gap through addition of the correct nucleotide and restoration of DNA integrity by sealing the nick. DNA polymerase β (Pol β) is a key enzyme in the single-nucleotide BER pathway.3 It catalyzes the removal of 5′-deoxyribose phosphate (5′-dRP) generated after excision of the damaged base by DNA glycosylase and incision of the abasic site by apurinic/apyrimidinic (AP) endonuclease 1 (APE1). It also fills in the DNA gap with the corresponding complementary nucleotide.4-6 The product of these consecutive reactions is a repaired nicked DNA that subsequently undergoes ligation to complete restoration of the native DNA structure. Pol β also plays an essential role in long-patch BER pathway by conducting strand displacement synthesis and controlling the size of the excised flap.7,8 The dual function of Pol β is reflected in its two-domain architecture. Human Pol β, a singlechain polypeptide of 335 amino acids, folds into two discrete domains linked by a short proteasesensitive region (Figure 1A).4,9,10 Removal of the 5′-dRP moiety takes place entirely within the N-terminal 8 kDa lyase domain. Polymerase activity is confined within the C-terminally positioned 31 kDa domain and its three subdomains which participate in duplex DNA binding (D, fingers subdomain), polymerization (C, palm subdomain), and nascent base pair binding (N,

4


Page 4 of 51

Page 5 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

thumb subdomain), respectively. Both domains act synergistically to position the substrate optimally for sequential processing.5,12 The lyase domain (Figure 1B) folds in a stable bundle of four alpha helices (I – amino acids 15-26; II – amino acids 36-47; III – amino acids 56-61; and IV – amino acids 69-78) packed as two antiparallel pairs with 50-60o crossing between the pairs. The III/IV set of helices forms a helix-hairpin-helix (HhH) motif that binds DNA in a non-sequence-specific manner, via hydrogen bonds between protein backbone nitrogens and DNA phosphate groups.12,13 Helix II, the hairpin of HhH motif and the Ω-type loop (amino acids 27-35) connecting helices I and II display significant backbone dynamics responsible for adjusting lyase domain conformation for efficient binding to its DNA substrate. In contrast, lyase catalytic residues Lys35, Tyr39 and Lys72 show highly restricted backbone motions.14 Removal of the dRP group from AP endonuclease-incised abasic site is likely the rate-limiting step for single-nucleotide BER.15 Several amino acid residues make key contributions to the Pol β-catalyzed lyase reaction (Fig. 1A). Glu26, Ser30 and Glu71 are involved in sugar deprotonation at C2′.12 His34 participates in base-stacking interactions with the downstream template base of the gapped DNA substrate. Lys35 plays a key role in sugar ring opening and, along with Lys 60 and Lys68, is involved in 5′-PO4 binding.5,12 Tyr39 stabilizes the deprotonated form of catalytically important Lys7216 and maintains proper folding of the whole 8 kDa domain.17 Thr79 is part of a hydrogen bonding network within the HhH motifs that is important for optimal positioning of the gapped DNA substrate in the active site.18 The only residue known to be directly involved in catalysis is Lys72,17,19 although structural and modeling data suggest that Lys84 could serve as its alternative,16 and some other basic residue (e.g., the conjugate base of Glu26) may facilitate abstraction of C2′-H,16 leading to

5


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

removal of the 5′-dRP moiety by β-elimination (Figure 2).5 The intermediacy of a Schiff base between the ring-opened aldehydic tautomer of the 5′-dRP moiety and the ε-amino group of Lys72 has been verified by reduction with sodium borohydride.17 As expected, the lack of an amino group at position 72 severely compromises lyase activity. Thus, the Lys72Ala Pol β mutant retains less than 5% of wild-type activity for 5′-dRP excision.12,16 A similar effect is observed upon acetylation of Lys72 by p300 transcriptional coactivator20 or substitution of Lys72 with glutamine or arginine.16,17 We sought to explore more subtle alterations in the position and/or nucleophilicity of the catalytic amino group to minimize possible conformational perturbations of the lyase catalytic pocket. A range of lysine analogues (Figure 3) was synthesized and each was incorporated at position 72 of human Pol β by coupled in vitro transcription/translation in the presence of a chemically misacylated tRNA.21-28 Modified Pol β enzymes were characterized for their ability to form Schiff base complexes, perform 5′dRP removal and support BER. The results provide insight into the interplay between the positioning, pKa and nucleophilicity of the amino group which conduce to efficient Schiff base formation and resolution. They also define the boundaries of Lys72 flexibility and could, for example, be useful in developing selective screens for identifying Pol  inhibitors engaging the main lyase catalytic nucleophile for binding. EXPERIMENTAL PROCEDURES Chemical Synthesis of Lysyl-pdCpA Derivatives. The syntheses of lysine derivatives 1 − 6 and their pdCpA esters are outlined in Schemes 1 and S1−S5, respectively. Biochemical Experiments DNA Template. The coding sequence of H. sapiens Pol β (NCBI, Accession No NM_002690) was codon-optimized for expression in E. coli (codon adaptation index increase from 0.63 to

6


Page 6 of 51

Page 7 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

0.89), and cloned as a NcoI/XhoI insert into pET28b expression vector (Novagen), resulting in the C-terminal placement of a hexahistidine tag. Small-scale plasmid DNA isolation was performed with a GenElute Plasmid Miniprep Kit (Sigma). The codon for Lys72 (AAG) was converted to an amber stop codon (TAG) with a QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) using primer 5′-GGCACCAAAATCGCAG AATAGATCGATGAATTTCTGGCTACGGG-3′ and its complementary strand. Maxipreparations of mutagenized and wild-type pET28b:Pol β vectors for in vitro protein synthesis were done using a E.Z.N.A. Plasmid DNA Maxi Kit (Omega Bio-tek). DNA samples were additionally subjected to extraction with 25:24:1 phenol‒chloroform‒isoamyl achohol, buffered with Tris at pH 8.0, and then treated with ethanol to effect precipitation. The DNA was then resuspended in RNase-free water to a final concentration of 1 mg/mL. Preparation of E. coli S−30 Extract. A single fresh colony of BL21(DE3) E. coli was grown in 3 mL of LB medium for 3 h at 37 oC and 190 rpm and used to inoculate 200 mL of LB supplemented with 0.5 mM IPTG. The culture was further incubated at 37 oC and 190 rpm until an OD600 of 0.8-0.9 was reached. Cells were collected by centrifugation at 3500  g for 15 min at 4 oC and weighed. The cells were then washed three times with 20 mL/g of S30A buffer (14 mM MgOAc, 60 mM KOAc, 10 mM Tris-OAc, pH 8.2, containing 10 mM DTT and 0.5 mL/L of βmercaptoethanol), once with 10 mL/g of S30B buffer (same as S30A but with 50 L/L of βmercaptoethanol), and then resuspended in 1.27 mL/g of S30C buffer (S30A buffer without βmercaptoethanol). Every 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 phosphoenol pyruvate-K salt, 4 mM DTT, 0.048 mM 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

7


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 15,000  g and 4 oC for 1 h. The supernatants were carefully transferred to fresh tubes and stored in small aliquots at ‒80 oC until use. Transcription and Purification of tRNACUA-COH, and Ligation to Pentenoylprotected aminoacyl-pdCpA. Yeast tRNAPheCUA-COH transcript was generated with Ampliscribe T7 transcription kit (Epicentre Biotechnologies) from FokI-digested pYRNA8 DNA and purified by DEAE Sepharose CL-6B chromatography. Elution was carried out with a 0.1 ‒ 0.7 M step gradient of NaCl in 100 mM NaOAc buffer, pH 5.0. The collected fractions were precipitated with isopropanol, and the pellets were redissolved in RNAse-free water and analyzed by electrophoresis on 8% acidic (100 mM NaOAc, pH 5.0) acrylamide gels containing 7 M urea after staining with methylene blue. Ligation to the aminoacyl-pdCpAs was performed in a volume of 100 L at 37 oC for 1.5 h using 1 U/μL of T4 RNA ligase 1 (New England Biolabs), 20 L of ligation buffer (50 mM Hepes buffer, pH 7.5, containing 15 mM MgCl2), 1 μg/μL of tRNACUA-COH, 0.005 OD260/μL of aminoacyl-pdCpA and 15% (v/v) DMSO. Samples were precipitated with EtOH, resuspended in water and deprotected by treatment with 12.5 mM iodine at room temperature for 15 min. 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 acid 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

8


Page 8 of 51

Page 9 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

mM K-glutamic acid, 75 mM ammonium acetate, 20 mM MgOAc•4H20, 63 mM phosphoenol pyruvate- K salt, 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 (40U/µL), 0.1 µL of 100 protease inhibitor cocktail (Roche), 0.2 µL of 100 mg/mL rifampicin, 2.6 µL of E. coli S-30 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 depending on the suppression yield. Controls with tRNACUA-COH were set up 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. At the end of the run, 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 (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 as follows: 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 using a Pierce desalting column (Thermo Fisher Scientific). The sample was mixed with Talon resin (Clontech) equilibrated with the same buffer in 5:1 (v/v) ratio and rotated at 4 oC for 1 h. The suspension was then 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. Bound proteins were eluted with 3 column

9


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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. LC-MS/MS and Protein Structure Verification. Human Pol  putatively containing homoarginine at position 72 was digested with MS-grade Lys C protease (Thermo Fisher Scientific) according to the manufacturer’s recommendations. LC-MS/MS analysis was carried out using a LTQ Orbitrap Velos mass spectrometer equipped with an Advion nanomate ESI source, following a ZipTip (Millipore) C18 sample clean-up according to the manufacturer’s instructions. Peptides were eluted from a C18 precolumn (100 M id  2 cm, Thermo Fisher Scientific) onto an analytical column (75 M id  10 cm, Thermo Fisher Scientific) initially using 2% solvent B (acetonitrile, 0.1% formic acid) in solvent A (0.1% aqueous formic acid) for 5 min; followed by a 2 → 10% linear gradient of solvent B in solvent A over a period of 5 min; followed by a 10 → 35% linear gradient of solvent B in solvent A over a period of 35 min; followed by a 35 → 50% linear gradient of solvent B in solvent A over a period of 20 min; followed by a 50 → 95% linear gradient of solvent B in solvent A over a period of 5 min; followed by 95% solvent B in solvent A for 5 min; followed by a return to 2% solvent B in solvent A within 0.1 min and finally elution with 2% solvent B in solvent A for an additional 9.9 min. All flow rates were 400 nL/min. Data-dependent scanning was performed with Xcalibur v2.1.0 software using a survey mass scan at 60,000 resolution in the Orbitrap analyzer scanning mass/charge (m/z) 400-1600, followed by collision-induced dissociation tandem mass spectrometry (MS/MS) of the 14 most intense ions in the linear ion trap analyzer. Precursor

10


Page 10 of 51

Page 11 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ions were selected by the monoisotopic precursor selection setting with selection or rejection of ions held to a ± 10 ppm window. Dynamic exclusion was set to place any selected m/z on an exclusion list for 45 sec after a single MS/MS. Tandem mass spectra were searched against human reviewed proteins (downloaded from UniProtKB on 10/06/2015) using Thermo Proteome Discoverer 1.3 (Thermo Fisher Scientific) considering Lys C peptides with up to 2 missed cleavage sites. Modifications considered during the search included methionine oxidation (15.995 Da), cysteine carbamidomethylation (57.021 Da), and lysine to homoarginine substitution (42.0218 Da). Results from peptide identification were visualized with Scaffold v3.6.1 (Proteome Software Inc.). Proteins were accepted if they passed a minimum of two peptides identified at 95% peptide confidence and 99.9% protein confidence by the Peptide and Protein Profit algorithms, respectively. This work was carried out in the Analytical/Biological Mass Spectrometry Core at the University of Arizona under the direction of Dr. George Tsaprailis. Preparation of 5′-dRP−Labeled Substrate. The 34-mer oligodeoxyribonucleotide containing uridine at position 16 (5′-CTGCAGCTGATGCGCUGTACGGATGCCCGGGTAC3′) was 3′-end labeled by terminal transferase (Roche Life Science) using 3′-[α-32P] cordycepin 5′-triphosphate (Perkin Elmer) according to a protocol provided by the manufacturer. 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 and cooling slowly to 25 oC. To prepare the substrate for the enzyme assays by removing the the uracil base and cleaving 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) 11


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 a 30-μL volume with 25 nM enzyme and 40 nM substrate in 30 mM Hepes buffer, pH 7.5, containing 25 mM KCl, 5 mM MgCl2, and 1 mM DTT. Aliquots of 9 μL were taken at 1, 15 and 30 min and mixed with 1 μL 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-L 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. For the BER assay, typically carried out in a 15-μL incubation volume, 50 nM 5′-dRP DNA substrate and 15 nM enzyme were incubated for 10 min at 37 oC, with T4 DNA ligase (NEB) in 50 mM Hepes buffer, pH 7.8, supplemented with 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. 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. Trapping of Schiff Base Intermediates. Trapping of the DNA substrate−enzyme complex before 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

12


Page 12 of 51

Page 13 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

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 and fixed and processed as described above. RESULTS Preparation of tRNACUAs Activated with Lysine Derivatives. Lysine and five lysine analogues (1 – 6) were used to activate a suppressor tRNACUA transcript for the introduction of unnatural amino acids into position 72 of human DNA Pol (Figure1). As illustrated in Scheme 1 for lysine itself, the amino acid was protected as its bis-N-pentenoyl derivative (8) in 94% yield and then activated as the respective cyanomethyl ester (9). Admixture with pdCpA26 in DMF containing trimethylamine then provided bis-N-pentenoyllysyl-pdCpA. Finally, incubation with an abbreviated suppressor tRNA transcript (tRNACUA-COH) in the presence of T4 RNA + ATP gave bis-N-pentenoyllysyl-tRNACUA (Scheme 1).29,30 Also prepared analogously were suppressor tRNACUAs activated with thialysine (Scheme S1), piperidinylalanine (Scheme S2), methyllysine (Scheme S3), homoarginine (Scheme S4) and homolysine (Scheme S5). The product of each ligation reaction was analyzed on an acidic (pH 5.0) 8% acrylamide gel containing 7 M urea (Figure 4A).31 As illustrated previously,31 the aminoacylated tRNACUAs exhibited a lesser mobility than the abbreviated tRNACUA-COH used as a substrate for the ligase reaction. Synthesis and Lyase Assay of Modified Polymerases  Containing Lysine Derivatives at Position 72. Admixture of each of the activated suppressor tRNAs to a transcription-translation reaction programmed with the modified DNA Polgene containing a 13


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TAG codon at the position corresponding to position 72 in the elaborated protein, resulted in suppression of the UAG codon in the mRNA and the appearance of full length Pol  (Figure 4B). The suppression yields ranged from 16% for the modified Pol  containing N-methyllysine at position 72 to 100% for the protein containing homoarginine. Recombinant proteins were designed with a hexahistidine fusion peptide at their C-termini, thus enabling purification by IMAC, which was performed on Talon resin to minimize contaminant carryover. As exemplified by the purification of homoarginine-substituted Pol , modified enzyme typically appeared as the most prominent band on a Coomassie-stained gel and the only band detected by autoradiography (Figure 5A). The protocol for in vitro protein synthesis in the presence of chemically misacylated tRNA developed and used routinely in our laboratory produces a negligible level of misincorporation in the absence of misacylated tRNA (Figure 4B, control lane). A good illustration of the reliability of the protocol is the confirmation of Lys72homoArg substitution by mass spectrometry. The substitution would eliminate a Lys C cleavage site, as homoarginine is not a substrate for Lys C,32 consequently generating a signature peptide with a monoisotopic mass of 1475.80 Da that spans residues 69-81 of human Pol . This peptide, along with 25 other exclusive unique peptides (corresponding to 75% protein coverage) (Figure 5B) was identified experimentally and the presence of homoarginine at position 72 was confirmed by MS/MS sequencing (Figure 5C, D). Each of the purified proteins was assayed for its ability to mediate the lyase reaction of Pol , i.e. the removal of a dRP lesion from a DNA oligonucleotide that had an excision produced by APE1 at the phosphate group attached to the 5′-position of the UDG-generated apurinic acid lesion (Figure 2). As shown in Figure 6A, the removal of the dRP lesion alters the mobility of the

14


Page 14 of 51

Page 15 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

DNA oligonucleotide substrate, permitting the progress of the reaction to be monitored conveniently. Comparison of the lyase activities of the modified Pol  enzymes are illustrated in Figure 6B. While the enzymes containing thialysine (2), methyllysine (4) and homolysine (6) were clearly reasonably active in comparison with wild type, those containing piperidinylalanine (3) and homoarginine (5) seemed much less active. These conclusions were reinforced by an additional experiment in which (1:10 and 1:50) serial dilutions of the six enzymes were studied (Figure 6C). Trapping of the Pol -DNA Schiff Base Complex by Treatment with NaBH4. The imine functionality formed between the side chain NH2 group of Lys72 and the aldehydic Catom of the dRP lesion can be reduced readily by treatment with sodium borohydride,17 and this provides a convenient method to quantify the amount of Schiff base complex present. In the presence of excess DNA substrate, the enzyme will work at its maximum rate; therefore, the amount of formed intermediate would serve as a good surrogate for the avidity of the enzyme for the substrate. The 3′-32P end labeled UDG/APE1-processed DNA substrate and Pol  were incubated on ice in the presence of 20 mM NaBH4, then aliquots were taken at predetermined times from 10 seconds to 10 minutes. The aliquots were added to an SDS loading dye and analyzed by SDS−PAGE. As shown in Figure 7, in addition to the wild-type Pol , strong bands at the position expected for the crosslinked protein−DNA product were evident in the reaction mixtures containing enzymes with thLys (2) and mLys (4), and to a lesser extent hLys (6). For all of these reactions, the E-S complex formed very quickly, precluding calculation of the reaction rates. In contrast, the enzymes containing pipAla (3) or hArg (5) demonstrated only limited ability to form the intermediate.

15


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The kinetics of Schiff base formation from the Pol  construct containing hArg (5) was analyzed more carefully. As shown in Figure 8, under the experimental conditions used the appearance of the intermediate did increase as a function of time, and seemed largely complete within 5 minutes. From the plotted graph depicting the dynamics of generated product over the monitored time period, the apparent rate of Schiff base formation was calculated to be 0.00034 sec-1. However, it may be noted that in the absence of a protein−DNA Schiff base, the aldehydic Catom of the dRP lesion can likely also be reduced by NaBH4, thus obscuring a slower formation of the Schiff base complex to a finite extent. Thus, the amount of crosslinked product apparent in Figure 8 may understate the rate of Schiff base formation, especially for those constructs which form this intermediate relatively slowly. One additional experiment to monitor Schiff base formation was also carried out. In this case, samples of the DNA substrate and individual enzymes were mixed on ice, then treated with NaBH4 immediately following admixture, and also after 10 and 30 minutes. As shown in Figure 9, the earliest time points for the constructs containing Lys (1), thLys (2), mLys (4) and hLys (6) clearly contained the greatest amounts of trapped Schiff base products in each case, reflecting rapid Schiff base formation. The diminution of these bands observed following addition of NaBH4 after 10 or 30 minutes was likely due to conversion of the Schiff base intermediate to product from which the dRP lesion had been excised. Quantification in Figure 8B illustrates the rapid formation of Schiff base in the constructs containing amino acids 1, 2 4, and 6, but the absence of trapped Schiff base in the constructs containing 3 or 5 at position 72. Interestingly, while the construct containing mLys (4) at position 72 had the second strongest trapped band for the initial time point, this construct afforded the strongest band after 10 minutes of incubation. Plausibly, this may reflect slower excision of the dRP lesion by this enzyme (cf. Figure 2).

16


Page 16 of 51

Page 17 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

BER Assay with the Modified Polymerase  Enzymes. Also studied was the excision of dRP from the duplex substrate, and repair of the lesion via the actions of the Pol  polymerase activity and T4 DNA ligase (Figure 10). As shown in Figure 11A, the excision reaction proceeded to essentially the same extent when using Pol  prepared from in vitro translation of wild-type mRNA, as from the same enzyme prepared by in vitro suppression of a UAG codon at position 72 of the mRNA with lysyl-tRNACUA. The formation of “repaired”, full length DNA oligonucleotide was dependent both on the presence of DNA polymerase , and T4 DNA ligase (Figure 11B). The BER assay (Figure 10) was used to analyze the behavior of the modified Pol  proteins. The repaired DNA oligonucleotide resulting from the successive actions of the lyase and polymerase activities of Pol , as well as T4 DNA ligase, afforded repaired 34-nt products in the presence of modified Pol  constructs containing thLys (2), mLys (4) or hLys (6) (Figure 12). Consistent with their limited activity in the lyase reaction (Figures 5, 6 and 8), the enzymes containing pipAla (3) and hArg (5) afforded only limited amounts of repaired DNA oligonucleotide. These results are entirely consistent with the relative activities of the constructs in the initial lyase reaction. Modeling of the Modified Polymerases . In an insightful analysis of the mechanism of BER by DNA Pol , Prasad et al. noted that in an X-ray crystal structure of Pol  bound to a DNA duplex substrate, Lys72 was too far from the dRP lesion to allow the lyase reaction to proceed as envisioned (cf Figure 13A).16 Accordingly, they suggested a rotation of ~120o about the dRP 3′-phosphate group, which would position Lys72 appropriately in the anticipated catalytic binding site. Figure 13A illustrates the position of Lys 72 relative to the dRP lesion according to this model. The distances from Nε-C1′ and Nε-O4′ of the dRP moiety are 3.5 Å and 17


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.0 Å, respectively. The Lys72thLys mutant (Figure 13B), which has been found in this study to retain significant BER activity, had predicted Nε-C1′ and Nε-O4′ distances of 4.8 Å and 4.7 Å, respectively, according to this model. In comparison, the Lys72hArg mutant (Figure 13C), found to be very weakly active in the lyase and BER assays, was predicted by this model to have Nω1C1′ and Nω1-O4′ distances of 6.5 Å and 7.1 Å, respectively. DISCUSSION During natural selection, enzymes gradually adopt structures enabling binding and conversion of their substrates, thus leading to efficiency of catalysis relevant to the biological function. Small deviations from the evolutionary established protein fold may have a significant impact on enzyme activity, as exemplified by many studies in the field of rational protein design and directed evolution.37,38 Despite the broad range of experimental approaches and software developments for gene and protein diversification, these methods are not ideal for studying the sensitivity of the enzymatic reactions to small changes in the geometry and reactivity of active site residues as they are limited by the structural diversity of the proteinogenic amino acids. In the present study, we have employed unnatural amino acid incorporation by amber codon suppression in the presence of chemically aminoacylated tRNA as a strategy to explore the tolerance to subtle alterations of the main catalytic nucleophile in the lyase pocket of human DNA Pol . According to our current understanding, during the initial binding of the abasic DNA substrate to Pol  lyase domain undergoes significant backbone motions of the segments connecting helices 1 and 2, and helices 3 and 4. Following this event, the lyase catalytic pocket, bracketed by the side chains of amino acids Glu26, Ser30, Lys35, Tyr39, Lys68, Lys84 and the primary nucleophile Lys72, retains a relatively rigid conformation.14 Subsequent positional readjustment

18


Page 18 of 51

Page 19 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

of the sugar associated with the damaged base to bring C1′ to a catalytically relevant distance from Lys72 relies entirely on its own flexibility14,16 and does not significantly perturb the existing network of interactions among the important residues. Nε of Lys72 participates directly in the protonation of O4′ which induces sugar ring opening and enables a nucleophilic attack at C1′ resulting in formation of an imino intermediate (Schiff base) (Figure 2). Hydrogen bonding of Lys72 to the neighboring Tyr39 maintains the stability of the neutral Nε.16 The importance of this interaction is corroborated by the loss of the 5′-dRP excision ability of the Tyr39Gln mutant enzyme.17 The imine at C1′ lowers the pKa of C2′ thus facilitating water-mediated abstraction of the proton at C2′ with participation of Ser30-hydrogen bonded Glu26. This triggers scission of the C3′−O phosphodiester bond and removal of the 5′-dRP group from the Schiff base complex.16 A structural analysis of the lysine analogues (Table 1) used in our study in the context of the lyase reaction mechanism demonstrates that they all are protonated at physiological pH, therefore potentially capable of generating the open-chain aldehyde forms of the prevalent α- and β-hemiacetals of the AP site.43 However, compared to lysine itself, the pKas of their basic center is modulated, being slightly lower – due to heteroatom substitution in the side chain (thialysine) or elongation of the side chain by one methylene group (homolysine), or slightly higher – due to N-methylation (methyllysine), or including the amino group as a part of a heterocycle (piperidinylalanine) or guanidino group (homoarginine). The introduced structural alterations also alter the distance between Cα and the side-chain nucleophilic N, which at the level of free amino acids is in the range of ±1.3 Å from the original Lys72 Nε position. The initial value for the Cα-Nε distance of 6.36 Å remains the same only for methyllysine (Table 1).

19


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Taken together, the changes in position and basicity of the side chain N atoms in each lysine analogue relative to lysine are reflected in the characteristics noted for the individual modified enzymes. This includes the facility with which they form a Schiff base complex, the stability of the generated imine intermediate, and the efficiency of processing of the 5′‐dRP lesion. Single amino acid modification of the primary nucleophile in the lyase catalytic pocket of Pol is not expected to transmit structural alterations to the polymerase domain as evidenced by unaltered DNA binding affinity, recognition of 5′-phosphorylated gapped DNA substrate and DNA nucleotidyltransferase activity of the Lys72Ala or Lys72Arg mutant proteins.12 Therefore, the polymerase activities of the Pol enzymes modified with unnatural amino acids were not subjected to detailed study. However, lyase activity was assessed both separately and in the context of the BER reaction, and in all instances the results from both assays were comparable, confirming unaltered polymerase activity. Of all the modified enzymes analyzed in the present study, the methyllysine variant was closest to the wild-type protein in regard to its ability to form a Schiff base complex and remove the 5′dRP group. This undoubtedly reflects the unchanged position of the primary nucleophile in the lyase cavity. The observed reduction (by ~20%) in the lyase and BER activities may have resulted from a greater stability of the imine intermediate resulting from the electron-donating effect of the N-methyl group. The Cα-NΖ distance in free homolysine is 1.24 Å greater compared to the Cα-Nε distance in free lysine, which, in the context of the two Pol s containing these amino acids at position 72 translates into an altered position of the catalytic nucleophile relative to C1′ of the 5′-dRP lesion, and to the oxygen atom of the Tyr39-OH group. This, in combination with slightly decreased basicity, may form the basis for the observed lower rate of formation of the Schiff base complex

20


Page 20 of 51

Page 21 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

by homolysine-substituted Pol as compared with wild typeUnder the conditions of the assay, the modified enzyme required slightly longer time to reach a steady-state level of the intermediate, while the wild-type enzyme reached this point within 10 sec (Figure 6). The imine complex also demonstrated a somewhat lower rate of resolution, although this might be obscured by the slow formation rate. Overall, compared to control, BER activity dropped to about 35% (Figure 11). Two modified enzymes, those having piperidinylalanine or homoarginine at position 72, demonstrated minimal ability to form a Schiff base intermediate and repair the 5′-dRP lesion. A similar effect was detected upon replacement of Lys72 with arginine. The mutant protein had virtually no lyase activity because the intermediate was formed at below 5% of the control levels.17 We generated an in silico model of the Lys72Arg mutant enzyme which showed that at least one of the amino groups of the guanidinium moiety is positioned at a catalytically relevant distance from the 5′-dRP substrate (data not shown). Therefore, the formation of the Schiff base complex appears to be less favorable than for the enzyme having Lys 72. At physiological pH, the guanidinium group of arginine exists almost entirely in the protonated form with a pKa of 12.48. The higher pKa of the guanidinium cation negatively affects its ability to protonate O4′ of the dRP group efficiently. This impediment can no doubt be extrapolated to hArg (5) which, in addition, is unfavorably positioned relative to the 5′-dRP lesion (Figure 13C). Collectively, the above effects result in the observed slow rate of Schiff base formation for the modified enzyme. The determined value of 0.00034 s-1 for Schiff base formation is more than two orders of magnitude lower compared to the corresponding value of 0.074 s-1 for the Leu22Pro cancer variant of Pol that has very low 5′-dRP lyase activity.44 We were unable to find reference data for the wild-type enzyme.

21


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

It is likely that positional constraints can rationalize the impaired ability of pipAlasubstituted enzyme to form the intermediate, since aliphatic secondary amines have good nucleophilic properties. In effect, the unfavorable geometry of both hArg (5) and pipAla (3) preclude their ability to act as nucleophiles. Experimental data suggest that in such cases other nearby lysine residues, specifically Lys84, may act as an alternative nucleophile.12,17,19 From that perspective, we cannot exclude the possibility that the basal levels of the formed intermediate for hArg (5) and pipAla (3)-modified enzymes could result, at least in part, from a Lys84-substrate Schiff base complex. Thialysine closely resembles lysine in its structure. At the level of the free amino acids, there is a slight geometrical change due to longer Cβ–Sγ and Sγ–Cδ bonds compared to the corresponding of Cβ–Cγ and Cγ–Cδ bonds (1.82 Å and 1.81 Å vs 1.53 Å and 1.54 Å) and more acute Cβ–Sγ–Cδ angle compared to the Cβ–Cγ–Cδ angle (102.9° vs 110.0°).44,45 This would extrapolate to an increase in Cα-Nε distance by only 0.24 Å. However, in a protein context, according to the in silico model built using the most probable thialysine rotamer, the Nε-C1′ and Nε-O4′ distances are altered more drastically. They changed from 3.5 Å and 3.0 Å in wild-type Pol to 4.8 Å and 4.7 Å, respectively, in the modified enzyme. At the same time, the crucial Lys72 Nε – Tyr 39 O(OH) distance of 4.5 Å was increased by the modest 0.2 Å. These effects could be interpreted from the prospective of thialysine preference to a gauche C-S-C torsion angle whereas lysine preference is to an anti C-C-C torsion angle.46 In addition, the presence of the sulfide moiety in thialysine attenuates the pKa of the side chain nucleophile by approximately 1 pH unit. Taken together, these effects might explain decreased fitness of the modified enzyme containing thialysine in regard to Schiff base formation and repair of the 5′-dRP lesion. CONCLUSIONS

22


Page 22 of 51

Page 23 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

DNA Pol β originates from ancient Bacillus polymerase X and retains remarkable similarity in size and domain architecture across the animal kingdom from phylum Cnidaria to phylum Chordata.48,49 The physical association of polymerase and lyase activities within a single protein moiety enhances tremendously the efficiency of BER. The lyase reaction takes place in a lysinerich catalytic pocket where the removal of the dRP group via a β-elimination is accomplished by sophisticated network of interactions among several amino acid residues. As their mobility following the initial binding to the DNA substrate is relatively restricted,13 the optimal geometry for catalysis is achieved mostly at the expense of dRP flexibility.15 A key event for lyase reaction is conversion of Lys72 (pKa 10.5 ± 1.1)50 to a potent nucleophile through deprotonation, which depends on its proximity to O4′ of dRP as a proton acceptor and to Tyr39 for stabilization.15 In this context, even slight repositioning of the ε-amino group of the highly conserved49 catalytic Lys72 and/or alteration of its nucleophile strength could impact the efficiency of Schiff base formation and/or the resolution of the imine intermediate. Our data from characterization of a panel of human Pol β enzymes containing different lysine analogues at position 72 corroborate the above conclusions. We have demonstrated that the lyase/BER activity of the modified enzymes decreases in the order mLys > thLys > hLys > pipAla > hArg. The lesser formation of 5-phosphorylated gapped DNA for the mLys-substituted Pol  is mainly due to slower excision of the dRP lesion. For the thLys-modified enzyme, suboptimal Nε-O4′ distance along with slight change in 72 Nε nucleophilicity likely account for slower formation of the Schiff base intermediate. Plausibly, effects of the S atom on the microenvironment of the lyase catalytic pocket may also influence the facility of reaction. The reduced lyase/BER activity of hLys-Pol β appears to reflect a less favorable position of the amino group of the main catalytic nucleophile for protonation of O4′ and subsequent nucleophilic attack at C1′. The 23


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

geometries of pipAla (3) and hArg (5) likely exclude interaction with the 5′-dRP lesion, thus almost completely preventing Schiff base formation. ASSOCIATED CONTENT Supporting Information Procedures for the synthesis and characterization of lysine analogues and their N-protected pdCpA derivatives. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Phone (480) 965-6625. Fax: (480) 965-0038. E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Dr. George Tsaprailis, University of Arizona, for assistance with mass spectrometric protein structure characterization. ABBREVIATIONS Pol , DNA polymerase β; BER, base excision repair; dRP, 5'-deoxyribose phosphate; APE1, apurinic/apyrimidinic (AP) endonuclease 1; HhH, helix-hairpin-helix; IMAC, immobilized metal affinity chromatography; UDG, uracil-DNA glycosylase.

24


Page 24 of 51

Page 25 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

REFERENCES (1) Robertson, A. B., Klungland, A., Rognes, T., and Leiros, I. (2009) DNA repair in mammalian cells: Base excision repair: the long and short of it. Cell Mol. Life Sci. 66, 981–993. (2) Forsina, G., Fortini, P., Rossi, O., Carrozzino, F., Raspaglio, G., Cox, L. S., Lane, D. P., Abbondandolo, A., and Doglitti, E. (1996) Two pathways for base excision repair in mammalian cells. Biochemistry 271, 9573–9578. (3) Sobol, R. W., Horton, J. K., Kühn, R., Gu, H., Singhal, R. K., Prasad, R., Rajewsky. K., and Wilson, S. H. (1996) Requirement of mammalian DNA polymerase-β in base-excision repair. Nature 379, 183–186. (4) Kumar, A., Widen, S. G., Williams, K. R., Kedar, P., Karpel, R. L., and Wilson, S. H. (1990) Studies of the domain structure of mammalian DNA polymerase β. Identification of a discrete template binding domain. J. Biol. Chem. 265, 2124–2131. (5) Matsumoto, Y., and Kim, K. (1995) Excision of deoxyribose phosphate residues by DNA polymerase β during DNA repair. Science 269, 699–702. (6) Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., and Kraut, J. (1996) Crystal structures of human DNA polymerase β complexed with DNA: implications for catalytic mechanism, processivity, and fidelity. Biochemistry 35, 12742–12761. (7) Klungland, A., and Lindahl, T. (1997) Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1). EMBO J. 16, 3341–3348. (8) Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V. A. (1999) Role of DNA polymerase β in the excision step of long patch mammalian base excision repair. J. Biol. Chem. 274, 13741– 13743.

25


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(9) Kim, S.-J., Lewis, M. S., Knutson, J. R., Porter, D. K., Kumar, A., and Wilson, S. H. (1994) Characterization of the tryptophan fluorescence and hydrodynamic properties of rat DNA polymerase β. J. Mol. Biol. 244, 224–235. (10) Sawaya, M. R., Pelletier, H., Kumar, A., Wilson S. H., and Kraut, J. (1994) Crystal structure of rat DNA polymerase β: evidence for a common polymerase mechanism. Science 264, 1930–1935. (11) Sawaya, M. R., Prasad, R., Wilson, S. H., Kraut, K., and Pelletier, H. (1997) Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism. Biochemistry 36, 11205−11215. (12) Prasad, R., Beard, W. A., Chyan, J. Y., Maciejewski, M. W., Mullen, G. P., Strauss, P. R., and Wilson, S. H. (1998) Functional analysis of the amino terminal 8 kDa-domain of DNA polymerase beta as revealed by site-directed mutagenesis. DNA binding and 5’ deoxyribose phosphate lyase activities. J. Biol. Chem. 273, 11121–11126. (13) Casas-Finet, J. R., Kumar, A., Morris, G., Wilson, S. H., and Karpel, R. L. (1991) Spectroscopic studies of the structural domains of mammalian DNA β-polymerase. J. Biol. Chem. 266, 19618–19625. (14) Maciejewski, M. W., Liu, D., Prasad, R., Wilson, S. H., and Mullen, G. P. (2000) Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase β. Correlation with DNA binding and dRP lyase activity. J. Mol. Biol. 296, 229–253. (15) Srivastava, D. K., Berg, B. J., Prasad, R., Molina, J. T., Beard, W. A., Tomkinson, A. E., and Wilson, S. H. (1998) Mammalian abasic site base excision repair. Identification of the reaction sequence and rate-determining steps. J. Biol. Chem. 273, 21203–21209.

26


Page 26 of 51

Page 27 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(16) Prasad, R., Batra, V. K., Yang, X. P., Krahn, J. M., Pedersen, L. C., Beard, W. A., and Wilson, S. H. (2005) Structural insight into the DNA polymerase β deoxyribose phosphate lyase mechanism. DNA Repair 4, 1347–1357. (17) Matsumoto, Y., Kim, K., Katz, D. S., and Feng, J. A. (1998) Catalytic center of DNA polymerase β for excision of deoxyribose phosphate groups. Biochemistry 37, 6456–6464. (18) Maitra, M., Gudzelak, A., Jr., Li, S. X., Matsumoto, Y., Eckert, K. A., Jager, J., and Sweasy, J. B. (2002) Threonine 79 is a hinge residue that governs the fidelity of DNA polymerase β by helping to position the DNA within the active site. J. Biol. Chem. 277, 35550– 35560. (19) Deterding, L. J., Prasad, R., Mullen, G. P., Wilson, S. H., and Tomer, K. B. (2000) Mapping of the 5′-2-deoxyribose-5-phosphate lyase active site in DNA polymerase β by mass spectrometry. J. Biol. Chem. 275, 10463–10471. (20) Hasan, S., El-Andaloussi, N., Hardeland, U., Hassa, P. O., Burki, C., Imhof, R., Schar, P., and Hottiger, M. O. (2002) Acetylation regulates the DNA end-trimming activity of DNA polymerase β. Mol. Cell 10, 1213–1222. (21) Hecht, S. M., Alford, B. L., Kuroda, Y., and Kitano, S. (1978) “Chemical aminoacylation” of tRNAs. J. Biol. Chem. 253, 4517–4520. (22) Heckler, T. G., Chang, L. H., Zama, Y., Naka, T., and Hecht, S. M. (1984) Preparation of 2’(3’)-O-acyl-pCpA derivatives as substrates for T4 RNA ligase-mediated “chemical aminoacylation”. Tetrahedron 40, 87–94. (23) Heckler, T. G., Roesser, J. R., Xu, C., Chang, P.-I., and Hecht, S. M. (1988) Ribosomal binding and dipeptide formation by misacylated tRNAPhe’s. Biochemistry 27, 7254–7262.

27


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(24) Roesser, J. R., Xu, C., Payne, R. C., Surratt, C. K., and Hecht, S. M. (1989) Preparation of misacylated aminoacyl-tRNAPhe's useful as probes of the ribosomal acceptor site. Biochemistry 28, 5185–5195. (25) Bain, J. D., Diala, E. S., Glabe, C. G., Dix, T. A., and Chamberlin, A. R. (1989) Biosynthetic site-specific incorporation of a non-natural amino acid into a polypeptide. J. Am. Chem. Soc. 111, 8013–8014. (26) Robertson, S. A., Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., and Schultz, P. G. (1989) The use of 5'-phospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA. Nucleic Acids Res. 17, 9649–9660. (27) Robertson, S. A., Ellman, J. A., and Schultz, P. G. (1991) A general and efficient route for chemical aminoacylation of transfer-RNAs. J. Am. Chem. Soc. 113, 2722–2729. (28) Eisenhauer, B. M., and Hecht, S. M. (2002) Site-specific incorporation of (aminooxy)acetic acid into proteins. Biochemistry, 41, 11472–11478. (29) Lodder, M., Golovine, S., and Hecht, S. M. (1997) A chemical deprotection strategy for the elaboration of misacylated transfer RNA’s. J. Org. Chem., 62, 778–779. (30) Lodder, M., Golovine, S., Laikhter, A. L., Karginov, V. A., and Hecht, S. M. (1998) Misacylated transfer RNAs having a chemically removable protecting group. J. Org. Chem., 63, 794–803. (31) Varshney, U., Lee, C. P., and RajBhandary, U. L. (1991) Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase. J. Biol. Chem. 266, 24712−24718.

28


Page 28 of 51

Page 29 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(32) Knight, Z. A., Schilling, B., Row, R. H., Kenski, D. M., Gibson, B. W., and Shokat, K. M. (2003) Phosphospecific proteolysis for mapping sites of protein phosphorylation. Nat. Biotechnol. 21, 1047−1054. (33) Cherepanov, A. V., and de Vries, S. (2003) Kinetics and thermodynamics of nick sealing by T4 DNA ligase. Eur. J. Biochem. 270, 4315−4325. (34) Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. (2004) UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 13, 1605−1612. (35) Dunbrack, R. L. Jr. (2002) Rotamer libraries in the 21st century. Curr. Opin. Struct. Biol. 12, 431−440. (36) Gfeller, D., Michielin, O., and Zoete, V. (2012) Expanding molecular modeling and design tools to non-natural sidechains. J. Compt. Chem. 33, 1525−1535. (37) Steiner, K., and Helmut, S. (2012) Recent advances in rational approaches for enzyme engineering. Comput. Struct. Biotechnol. J. 2, e201209010. (38) Chen, T., and Romesberg, F.E. (2014) Directed polymerase evolution. FEBS Letters 588, 219−229. (39) Bolton, E. E., Chen, J., Kim, S., Han, L., He, S., Shi, W., Simonyan, V., Sun, Y., Thiessen, P. A., Wang, J., Yu, B., Zhang, J., and Bryant, S. H. (2011) PubChem3D: a new resource for scientists. J. Cheminform. 3, 32. (40) Hermann, V. P., and Lemke, K. (1968) Ionization constants and stability constants of copper (II) complexes of some amino acids and their sulfur-containing analogs. Hoppe-Seyler’s Z. Physiol. Chem. 349, 390−394.

29


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(41) Kennedy, K. J., Lundquist IV, J. T., Simandan,T. L., Kokko, K. P., Beeson., C. C., and Dix, T. A. (2000) Design rationale, synthesis, and characterization of non-natural analogs of the cationic amino acids arginine and lysine. J. Peptide Res. 55, 348−358. (42) Gfeller, D., Michielin, O., and Zoete, V. (2013) SwissSidechain: a molecular and structural database of non-natural sidechains. Nucleic Acids Res. 41, D327−D332. (43) Wilde, J. A., Bolton, P. H., Mazumder. A., Manoharan, M., and Gerlt, J. A. (1989) Characterization of the equilibrating forms of the aldehydic abasic site in Duplex DNA by 17O NMR J. Am. Chem. Soc. 111, 1894−1896. (44) Dalal, S., Chikova, A., Jaeger, J., and Sweasy, J. B. (2008) The Leu22Pro tumorassociated variant of DNA polymerase beta is dRP lyase deficient. Nucleic Acid Res. 36, 411−422. (45) Wright, D. A., and Marsh, R. E. (1962) The crystal structure of l-lysine monohydrochloride dehydrate. Acta Crystallogr. 15, 54−64. (46) Ammon, H. L., Prasad, S. M., and Gerlt, J. A. (1991) Structure of thialysine hydrochloride. Acta Crystallogr. Sect. C Cryst. Struct. Commun. 47, 1476−1478. (47) Messmore, J. M., Fuchs, D. N., and Raines, R. T. (1995) Ribonuclease A: Revealing structure:function relationships with semisynthesis. J. Amer. Chem. Soc. 117, 8057−8060. (48) Kodera, H., Takeuchi, R., Uchiyama, Y., Takakusagi, Y., Iwabata, K., Miwa, H., Hanzawa, N., Sugawara, F., and Sakaguchi, K. (2011) Characterization of marine X-family DNA polymerases and comparative analysis of base excision repair proteins. Biochem. Biophys. Res. Commun. 415, 193−199. (49) Bienstock, R. J., Beard, W. A., and Wilson, S. H. (2014) Phylogenetic analysis and evolutionary origin of DNA polymerase X-family members. DNA Repair 22, 77–88. 30


Page 30 of 51

Page 31 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

(50) Grimsley, G. R., Scholtz, J. M., and Pace, C. N. (2009) A summary of the measured pK values of the ionizable groups in folded proteins. Protein Sci. 18, 247–251.

31


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 51

___________________________________________________________ Table 1. Lysine Analogues Incorporated into Position 72 of Human DNA Pol  amino acid

distance from Cα to N (Å)a

pKa (side chain)b

L-lysine

6.36

10.44c

L-thialysine

6.6

9.38

L-piperidinylalanine

5.08

11.28d

L-methyllysine

6.36

10.82

L-homoarginine

5.3, 7.22, 7.54

12.23

L-homolysine

7.6

10.20

L-arginine

4.39, 6.48, 6.54

12.48c

__________________________________________________________ Analysis performed using PubChem 3D Viewer v2.0.39 bReferences 40‒42. Proteinogenic amino acids used as positive and negative controls are in boldface and italics, respectively. dpKa value for piperidine. a c

32


Page 33 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Legends to Figures

Figure 1. Domain and subdomain organization of DNA Pol β. (A) 3D-structure of 8 kDa lyase domain (light blue) and 31 kDa polymerase domain (dark green) of human Pol  (PDB entry 1BPY, DNA structure has been removed to improve clarity).11 Key amino acid residues of the lyase catalytic pocket are colored in dark blue, the catalytic nucleophile Lys72 is colored in red. The schematic underneath outlines domain and subdomain borders. (B) Primary structure of lyase domain showing the positions of the α-helices, type II β-turn and HhH motif. Figure 2. Proposed mechanism for dRP excision mediated by the lyase domain of DNA Pol . Adapted from ref. 15, with some participating protein residues removed to improve clarity. Figure 3. Lysine analogues incorporated into position 72 of DNA Pol , shown as their Nprotected pdCpA esters. Figure 4. Generation of modified Pol enzymes. (A) Ligation of aminoacylated pdCpA derivatives to suppressor tRNA-COH was monitored by acidic PAGE (8% acrylamide gel containing 7 M urea) and methylene blue staining. (B) Incorporation of lysine analogues into position 72 by coupled in vitro transcription/translation monitored by SDS-PAGE (4% stacking gel, 15% separating gel) and autoradiography. Yields of synthesized full-size enzymes (~39 kDa) were estimated based on the band intensity readings. Abbreviations: Lys – lysine (1), thLys – thialysine (2), pipAla – piperidinylalanine (3), mLys – methyllysine (4), hArg – homoarginine (5) and hLys – homolysine (6). The suppression yields are shown at the bottom of panel B. Figure 5. Purification and characterization of hArg72-modified Pol β. (A) SDS−PAGE of the purified enzyme visualized by parallel Coomassie staining and autoradiography. (B) Experimental sequence coverage (75%, brown) after LC-MS/MS analysis of Lys C-digested

33


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

modified enzyme. (C) Identified ions (colored) after MS/MS fragmentation of the precursor ion containing modified residue 72. (D) Fragmentation spectrum related to (C) (colors correspond to those in (C)). Figure 6. Lyase assay with wild-type and modified Pol . (A) Principle of lyase assay illustrated with in vitro synthesized wild-type Pol Pol  removes dRP (214 Da) from the substrate (S), which results in the appearance of a band (product, P) with greater electrophoretic mobility. (B) Lyase assay with wild-type and modified Pol  enzymes performed as described in Materials and Methods. (C) Same assay as in panel B, performed for 10 min with different enzyme dilutions (1; 10; 1:50). Figure 7. Trapping of the Pol −DNA Schiff base complex by treatment with sodium borohydride. Reaction mixtures containing 3′-32P end labeled UDG/APE1-processed DNA substrate and Pol  were assembled on ice in the presence of 20 mM NaBH4. Aliquots were taken at predetermined times (10-600 sec), mixed with an equal volume of SDS loading dye (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.72 M β-mercaptoethanol, and 0.1% Orange G) and analyzed by SDS-PAGE (4% stacking gel, 15% separating gel) followed by autoradiography. Figure 8. Kinetics of Schiff base formation in the presence of Pol containing homoarginine (5) at position 72. Data for the plot were obtained from the experiment presented in Figure 6 for the same enzyme. The “100%” value is defined based on the maximum amount of Schiff base complex observed under the experimental conditions (i.e. after 300 seconds). The image above the graph is an enhanced version of the original image in Figure 6 used as a data source. Figure 9. Trapping of Schiff base complexes. (A) The DNA substrate and (modified) enzymes were mixed on ice. Aliquots were taken at 1, 10 and 30 min, and then mixed with NaBH4 to 20 34


Page 34 of 51

Page 35 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

mM final concentration. Reactions were incubated on ice for an additional 30 min to stabilize the product via imine reduction. After addition of SDS loading buffer, aliquots were run on a 15% polyacrylamide gel and the trapped polymerase−DNA complexes were visualized by autoradiography. (B) Time-dependent formation of Pol −DNA Schiff base complexes based on the band intensity readings from the data in panel A. Figure 10. BER assay. Mechanistic steps involved in the short patch repair of a DNA lesion by polymerase . Figure 11. Base excision repair (BER) assay with wild-type and modified Pol  enzymes. (A) BER assay with in vitro synthesized wild-type Pol  and Pol  obtained after incorporating lysine at position 72 during in vitro translation from 72TAG DNA template. (B) Principle of BER assay demonstrated with in vitro synthesized wild-type Pol . The 19-mer 5′-dRPcontaining 3′-32P end-labeled substrate (fifth lane from the left, gray arrow) obtained after subjecting the 34-mer U-containing DNA duplex (first lane) to treatment with UDG and APE1 converts into the recovered 34-mer 3′-32P end-labeled product (second lane, black arrow) only in the presence of Pol andDNA ligase (+ dNTP). In the absence of Pol orDNA ligase the BER reaction did not take place (third and fourth lanes). The upper band in lane 3 (white arrow) is attributed to the adenylated intermediate formed from the 5′-phosphate of the labeled oligonucleotide by the action of DNA ligase + ATP,33 which cannot be converted to ligated product in the absence of gap filling by DNA polymerase . Figure 12. (A) Typical BER assay with in vitro synthesized wild-type and modified Pol  enzymes. Abbreviations: C+, initial 34-mer 5′-dRP containing labeled DNA strand; C-, the 19mer labeled DNA strand generated from APE1-processed 34-mer 5′-dRP-containing duplex as a part of BER reaction mixture not containing Pol . Yields of the recovered BER products were 35


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

calculated based on the corresponding band intensity readings after autoradiography. (B) BER activity of wild-type and modified Pol  Figure 13. In silico predicted repositioning of the main catalytic nucleophile relative to the dRP group upon substitution of Lys72 of human Pol with lysine analogues. (A) According to currently accepted model, in a non-catalytic position, the dRP group is too distant from Nε of Lys72 as indicated by the crystal structure of human Polcomplexed with tetrahydrofuran (abasic site)-containing DNA substrate (PDB entry 2P66)15 and should undergo a ~120o rotation about the 3′-phosphate to allow protonation of O4′ and nucleophilic attack at C1′ of the dRP group (reduction of Nε-C1′ and Nε-O4′ distances from 10.1 Å and 10.3 Å to 3.5 Å and 3.0 Å, respectively). (B) Model of Lys72thLys mutant predicting Nε-C1′ and Nε-O4′ distances of 4.8 Å and 4.7 Å, respectively. (C) Model of Lys72hArg mutant predicting Nω1-C1′ and Nω1-O4′ distances of 6.5 Å and 7.1 Å, respectively. Models of the wild- type and modified Pol enzymes were produced with Chimera 1.9 software.34 The Dunbrack rotamer library35 or SwissSideChain database36 were used to select the highest probability rotamer of the corresponding natural or nonproteinogenic amino acid.

36


Page 36 of 51

Page 37 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 1

37


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2

O O P O O-

.. O

-

H 3 N+

Lys72

OH

OH

O O P O O-

OH 3'

O P O O-

5'

O O P O O-

-

-

OH O

N

H

OH

O

N

OH

O

5'

3'

O O P O O-

OH

O H 2N

OH

5'

O

H 2O

Glu26 3'

Lys72

HOOC

O P O

OH

Lys72

HOOC

O P O

Glu26

-

Glu26 3'

38

Lys72

3'

OH

OOC

O P O O

Lys72

-

-

OH

OHH O

O P O O-

5'

H 2N ..

O O P O O-

-

O

5'

Page 38 of 51


Page 39 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 3

NH 2 N O

N

-O P O -

O

O

O

OpdCpA

O

O

NH2 O

O P O O

N O

HN

N

N

NH

O

O

N

OH OH

HN

NH

O

OpdCpA

S

O

1

2

lysine (Lys)

thialysine (thLys)

pdCpA

O N

O

OpdCpA

O

OpdCpA

O

NH

O NH O

3 piperidinylalanine (pipAla)

H3CN

NH

O

H 2N

N H

NH O

O

5

6

homoarginine (hArg)

homolysine (hLys)

4

39

H N

NH

O

methyllysine (mLys)

OpdCpA

O

OpdCpA


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 51

Figure 4

40


Page 41 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 5

41


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 51

Figure 6

42


Page 43 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 7

43


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 51

Figure 8

44


Page 45 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 9

45


Biochemistry

Figure 10

P

A U C A G T G G T C

A C A G T G G T C

URACIL‐DNA GLYCOSYLASE

DNA‐POLYMERASE β

A C A G T G G T C APE1 ENDONUCLEASE

DNA‐POLYMERASE β

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 51

OH

P

A C C T G G LIGASE 3

C A G A T G G T C

A C C A G T G G T C

46

A G T C


Page 47 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 11

47


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 51

Figure 12

48


Page 49 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 13

49


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 51

Scheme 1. Synthetic Route Employed for the Preparation of N-Protected Lysyl-pdCpA (1) and Enzymatic Conversion to Lysyl-tRNACUA

O O O

N O

1:1 dIoxane-water Na 2CO3

O H2 N

OH

7

O

O

O

OH

N H

94%

NH 2

O

O

ClCH 2CN, Et3N, acetonitrile

NH 63%

8

O

CN

O

O

OpdCpA

pdCpA, Et3N, DMF N H

N H

NH 79%

9

O

NH

1

O

NH2 N

O

tRNA-CC

O P

1. T4 RNA ligase tRNA-COH

O

N

N N

O

O O

2. aq iodine

O

OH

NH 2 H 2N

lysyl-tRNACUA

50


Page 51 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

ToC Graphic


Probing the Flexibility of the Catalytic Nucleophile in the Lyase

Recommend Documents