©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Involvement of Glutamic Acid 23 in the Catalytic Mechanism of T4 Endonuclease V (*)

(Received for publication, August 17, 1994; and in revised form, November 28, 1994)

Raymond C. Manuel Katherine A. Latham M. L. Dodson R. Stephen Lloyd (§)

From the Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, Texas 77555

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Bacteriophage T4 endonuclease V has both pyrimidine dimer-specific DNA glycosylase and abasic (AP) lyase activities, which are sequential yet biochemically separable functions. Previous studies using chemical modification and site-directed mutagenesis techniques have shown that the catalytic activities are mediated through the alpha-amino group of the enzyme forming a covalent (imino) intermediate. However, in addition to the amino-terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 near the active site, and this residue has been strongly implicated in the reaction chemistry. In order to understand the role of Glu-23 in the reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, and the mutant proteins were evaluated for DNA glycosylase and AP lyase activities using defined substrates and specific in vitro and in vivo assays. Replacement of Glu-23 with Gln, Cys, or His completely abolished DNA glycosylase and AP lyase activities, while replacement with Asp retained negligible amounts of glycosylase activity, but retained near wild type levels of AP lyase activity. Gel shift assays revealed that all four mutant proteins can recognize and bind to thymine dimers. The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely to require an acidic group in the active site of the enzyme.


INTRODUCTION

Bacteriophage T4 endonuclease V (EC 3.1.25.1), encoded by the den V gene, initiates the removal of pyrimidine dimers in DNA and was the first DNA repair enzyme to have its structure solved by x-ray crystallography(1) . This enzyme is a DNA glycosylase with concomitant AP lyase activity, known to be specific for cis-syn cyclobutane pyrimidine dimers, although it has been reported to act on trans-syn-I dimers at extremely low efficiency(2) . Extensive studies have been performed to address various structural and functional aspects of the enzyme (reviewed in (3) and (4) ).

One of the questions concerning the reaction mechanism of endonuclease V that remained unanswered for many years was whether or not the same active center involved in the pyrimidine dimer N-glycosylase activity was also responsible for the AP (^1)lyase reaction. The N-glycosylase and AP lyase activities of endonuclease V have been shown to occur by a two-step process. First, the N-glycosylic bond of the 5`-pyrimidine of the dimer is cleaved(5, 6) . This is followed by the nicking of the phosphodiester bond between the two dimerized pyrimidines(7, 8) . Chemical modification studies of endonuclease V by reductive methylation have shown that the alpha-NH(2) group of the enzyme is involved in the chemical mechanism of both glycosylase and AP lyase activities(9) . The presence of a primary amine at the amino terminus is required for enzymatic function, but is not necessary for substrate recognition. Furthermore, the exact location of the alpha-amino group relative to the rest of the enzyme is critical for catalytic activity(10) .

The proposed reaction mechanism for endonuclease V displays the characteristics of a reaction with an imino enzyme-substrate intermediate. The existence of the imino intermediate has been demonstrated by the following criteria: (a) inhibition of the enzyme by cyanide in a concentration-dependent manner, as cyanide can react with the protein-DNA intermediate forming a reversible tetrahedral complex, (b) reduction of the imino intermediate to a covalent enzyme-substrate product by sodium borohydride (NaBH(4)) and subsequent isolation of the complex, and (c) demonstration that the amino-terminal alpha-amino group is directly involved in the formation of the imino intermediate(11) . These studies led to the formulation of our current understanding about the chemistry of the catalytic mechanism of T4 endonuclease V which is schematically illustrated in Fig. 1. A covalent imino intermediate is formed as a result of a nucleophilic attack by the alpha-NH(2) terminus at C-1` on the 5` sugar in the pyrimidine dimer (panel B). The phosphodiester bond is cleaved by beta-elimination facilitated by the removal of pro-S 2` hydrogen, resulting in an alpha,beta-unsaturated aldehyde and a 3`-phosphate as shown in panel D(12) . However, the enzyme can also dissociate without undergoing the beta-elimination reaction thus generating an AP site (panel C). It is believed that the concentration of the nucleophiles in the medium determines the rate of dissociation of the enzyme before beta-elimination.


Figure 1: Panel A is a schematic representation of the proposed reaction mechanism of T4 endonuclease V. Panel B illustrates the chemistry of the glycosylase step with a thymine dimer, a substrate for T4 endonuclease V. Panel C shows the dissociation of the covalent intermediate. Panel D depicts the actual lyase reaction, beginning with the protonated Schiff base intermediate.



The x-ray crystal structure of T4 endonuclease V (1) shows that the side chain of Glu-23 is positioned close to the enzyme's active site (alpha-NH(2) of T2). Previous site-directed mutagenesis studies of Glu-23 have further demonstrated its importance in enzyme catalysis such that E23Q was shown to completely abolish both glycosylase and AP lyase activity while E23D only abolished the glycosylase activity(13, 14) . Although the above studies have demonstrated the significance of Glu-23, mechanistic analyses have not been provided and the role of the alpha-NH(2) moiety was not taken into consideration for the formalization of the reaction mechanism. In this study, a series of mutants were constructed at Glu-23 to further probe into the biochemical mechanism of catalysis and address specific questions on the role of Glu-23 in the reaction mechanism.


EXPERIMENTAL PROCEDURES

Bacteria, Phage, and Plasmids

The Escherichia coli strains, M13 phage constructs, and plasmids used in this study are described in Table 1.



Oligonucleotide Site-directed Mutagenesis of the Endonuclease V Gene den V

All of the procedures for creating mutants of endonuclease V by site-directed mutagenesis have been previously described in detail elsewhere(15) . The sequence of the oligonucleotides used for mutagenesis are provided in Table 2.



Purification of Mutant Enzymes

The purification scheme of the mutant endonuclease V proteins is shown in Fig. SI. The mutant endonuclease V proteins were expressed off of the O(L)P(R) hybrid promoter in E. coli strain AB2480. Two-liter cultures were grown at 30 °C for 18 h in LB medium containing 100 µg/ml ampicillin. Cells were pelleted by centrifugation at 4000 times g at 4 °C and resuspended in 300 ml of Buffer A (20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 200 mM KCl, and 10% (v/v) ethylene glycol). Cells were disrupted by sonication, and the lysates were clarified by centrifugation at 8000 times g for 20 min at 4 °C. The supernatant was loaded onto a single-stranded DNA agarose column (80 ml) previously equilibrated with Buffer A. The mutant proteins were eluted with a linear gradient in which KCl concentration was increased from 0.2 M to 1.5 M (200 ml each). Fractions were monitored by immunoblot analysis and Coomassie Brilliant Blue staining of 15% SDS-PAGE gels. Fractions containing the mutant endonuclease V protein were pooled, and a saturated (NH(4))(2)SO(4) solution (4.1 M) was added until the final concentration approached 1 M. The sample was loaded onto a phenyl-Sepharose column (20 ml), previously equilibrated with Buffer B (25 mM NaH(2)PO(4) (pH 6.8), 1 M (NH(4))(2)SO(4), 1 mM EDTA, and 10% (v/v) ethylene glycol). After washing the column with buffer B, the proteins were eluted with a linear gradient of (NH(4))(2)SO(4) where the concentration was reduced from 1 M to 0 M. The flow-through and the gradient fractions were analyzed on SDS-PAGE gels, and endonuclease V protein was identified by immunoblot analysis and silver staining. The enzyme was further purified and concentrated by FPLC using a Mono S HR 10/10 (8-ml) column (Pharmacia Biotech). The samples were dialyzed against 2 changes of 25 mM NaH(2)PO(4) (pH 6.8), 1 mM EDTA, 100 mM KCl, and 10% (v/v) ethylene glycol and loaded onto the Mono S column, previously equilibrated with the dialysis buffer. The mutant endonuclease V proteins were eluted using a linear gradient of 0-1 M KCl. The endonuclease V mutant proteins eluted at 600 mM. The peak fractions were analyzed on 15% SDS-PAGE gels, and the concentration was determined by BCA (Pierce) assay.


Scheme I: Purification scheme of mutant endonuclease V proteins.



Amino Acid Sequencing of Mutant Enzymes

In addition to confirming the desired mutations by dideoxy sequencing of the mutant genes(16) , NH(2)-terminal sequence analysis of the mutant proteins was also performed to verify the mutations. The purified mutant proteins (5 µg) were separated on a 15% SDS-PAGE gel and electroblotted onto a polyvinylidene difluoride membrane (0.2 µ) at 50 V for 1 h at room temperature in a Bio-Rad Trans-Blot Cell, in CAPS buffer (pH 11) containing 10 mM CAPS and 10% (v/v) methanol. The PVDF membrane was stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 in 40% (v/v) methanol and 1% (v/v) acetic acid for 5 min and destained with 50% (v/v) methanol. The bands were excised and submitted to the Protein Chemistry Core, Sealy Center for Molecular Science, UTMB for sequence analysis.

Intracellular Accumulation of Endonuclease V Proteins

E. coli strain AB2480 harboring the expression vector pGX2608, pGX2608-den V, or the pGX2608 den V mutant constructs (E23H, E23C, E23Q, and E23D) were grown to stationary phase at 30 °C in LB medium containing 100 µg/ml ampicillin. The cells were harvested and crude cell lysates were analyzed for the accumulation of endonuclease V protein by SDS-PAGE followed by Western blot analysis(17) .

In Vivo Assay of Mutant Enzyme Activity

E. coli strain AB2480 harboring pGX2608-den V, pGX2608-den V, or the mutant constructs (pGX2608-den V E23H, E23C, E23Q, E23D) were grown to stationary phase at 30 °C in LB medium containing 100 µg/ml ampicillin. Survival was measured as a function of colony-forming ability relative to the unirradiated cells(17) .

Pyrimidine Dimer-specific Nicking Activity

Pyrimidine dimer-specific nicking assays on plasmids and synthetic oligonucleotides were performed as described previously(15) .

AP Lyase Activity of Endonculease V Mutants on Oligonucleotides

A 49-base oligonucleotide containing a single uracil at position 21 was obtained (Midland Research Co.) with the sequence: 5`-AGCTACCATGCCTGCACGAAUTAAGCAATTCGTAATCATGGTCATAGCT-3`. The oligonucleotide containing the uracil was 5`-end-labeled with [-P]ATP, annealed to its complementary strand, and then purified with NENsorb cartridges as per the supplier's instructions (DuPont). The radiolabeled oligonucleotide was dried in vacuum before resuspending in a buffer containing 70 mM HEPES (pH 8.0), 1 mM EDTA, and 1 mM 2-mercaptoethanol. This DNA was reacted with 1 unit of E. coli uracil DNA glycosylase (Epicentre Technologies) to create double-stranded DNA containing a site-specific AP site. Wild type and mutant endonuclease V proteins were incubated with the abasic site containing double-stranded DNA at 37 °C for 30 min. The reactions were terminated with loading buffer (95% formamide, 20 mM EDTA, 0.02% (w/v) bromphenol blue, 0.02% (w/v) xylene cyanol). The reaction products were separated by electrophoresis on 15% polyacrylamide gels, and the different bands were identified by autoradiography.

Binding of Mutant Endonuclease V Proteins to a cis-syn Thymine Dimer-containing Oligonucleotide

The 49-base oligonucleotide containing a site-specific cis-syn thymine dimer (0.39 ng) was 5`-end-labeled with [-P]ATP and T4 polynucleotide kinase, annealed to its complementary strand, and then allowed to bind with 200 ng of each of the endonuclease V mutants (E23H, E23C, E23Q, and E23D). Binding reactions were for 15 min at 20 °C in a buffer containing 0.5 times TBE, 5% (v/v) glycerol, 0.3 µg/ml poly(dI-dC)-poly(dI-dC), 150 mM NaCl, and 0.03% (w/v) bromphenol blue. The complexes were separated by electrophoresis in a nondenaturing polyacrylamide gel and subjected to autoradiography. In a separate experiment, different concentrations of the E23Q mutant or wild type protein were allowed to bind to the same substrate in the presence of either 100 mM NaCl or NaBH(4). The reaction conditions were the same as above except for the substitution of NaBH(4) for NaCl. NaBH(4) is known to covalently trap the wild type enzyme on the DNA.

Molecular Weight Determination of Endonuclease VbulletDNA Complexes

Binding of each of the mutant proteins to the 49-base pair oligonucleotide containing a site-specific thymine dimer resulted in two major complexes. The molecular weights of each of these bound complexes for the E23Q mutant, and thus the stoichiometry of binding, was determined by an adaptation of Ferguson analysis(^2)(18, 19) . The CS 49-mer was P-end-labeled using T4 polynucleotide kinase and then annealed to its complementary sequence. The E23Q mutant (1.2 µg) was reacted with 3.7 ng of this DNA in the binding reaction buffer (0.5 times TBE, 5% (v/v) glycerol, and 0.1% (w/v) bromphenol blue). The reaction was allowed to proceed for 30 min at 20 °C. The sample was then divided, and the DNA-protein complexes were separated by electrophoresis through six different nondenaturing polyacrylamide gels with acrylamide concentrations ranging from 4.5 to 9.0% (acrylamide:bisacrylamide, 19:1). Native protein standards (Nondenatured Protein Molecular Weight Marker Kit, MW-ND-500 Kit, Sigma) were also subjected to electrophoresis on each of the six gels. After electrophoresis, the gels were analyzed by autoradiography and then by Coomassie Blue staining. The relative mobility (R(f)) of each band was determined using the mobility of the bromphenol blue dye as the reference, and the data were analyzed as described previously(18, 19) .

Determination of the molecular weight of the complex formed between the E23Q mutant and an oligonucleotide containing a tetrahydrofuran (THF) residue was performed in the same manner. A 30-base oligonucleotide containing a synthetically produced THF residue (AP site analog) was provided by C. Iden, F. Johnson, and A. Grollman, SUNY, Stony Brook with the sequence 5`-ACCATGCCTGCACGAAXTAAGCAATTCGTA-3` where X is the furan residue(20) . This oligonucleotide (THF 30-mer) was P-labeled and annealed to its complementary strand. The E23Q mutant (1.2 µg) was allowed to bind to the radiolabeled double-stranded THF-containing 30-mer (0.34 µg) in the binding reaction buffer, and the complexes were subjected to electrophoresis and Ferguson analysis.


RESULTS

Introduction of Mutations at Glu-23 within the Endonuclease V Gene, den V

Previously, the den V structural gene had been subcloned into M13mp18 RF DNA. The single-stranded DNA from this phage was used as a template to engineer base changes in the den V gene such that the codon for Glu-23 was converted to codons for His-23, Cys-23, Gln-23, or Asp-23 (Table 1). The mutated den V structural genes were subcloned in the correct orientation into the expression vector pGX2608 at a unique ClaI restriction enzyme site, which is located immediately downstream of the hybrid phage promoter O(L)P(R). These plasmids were introduced into excision repair-deficient (uvrA) and recombination-deficient (recA) E. coli AB2480.

Accumulation of Mutant Endonuclease V Protein in E. coli Cells

Wild type and mutant forms of endonuclease V were expressed in repair-deficient E. coli, and the relative expression levels were compared to wild type. The proteins were electroblotted onto nitrocellulose membrane and detected by immunoblot analysis. Intracellular accumulation of each of the mutant enzymes was comparable to or even greater than wild type (Fig. 2A). This steady state accumulation of the respective proteins is important when the biological activity of the mutant enzymes is compared to that of the wild type.




Figure 2: A, Western blot analysis of mutant endonuclease V proteins accumulated in E. coli AB2480 expressing the plasmid-den V constructs. Samples containing 25 µg of total cellular protein were separated through a 15% SDS-PAGE gel and subjected to Western blot analysis. The mutant proteins and wild type were detected with a mouse anti-endonuclease V polyclonal antibody. The position of prestained molecular weight markers (Life Technologies Inc.) are indicated in the margin. Each mutant construct produced immunoreactive material that migrated at a position consistent with the known monomer molecular weight of endonuclease V. Lane 1, AB2480 pGX2608-den V-His-23; lane 2, AB2480 pGX2608-den V-Cys-23; lane 3, AB2480 pGX2608-den V-Gln-23; lane 4, AB2480 pGX2608-den V-Asp-23; lane 5, AB2480 pGX2608-den V; and lane 6, AB2480 pGX2608-den V. B, SDS-PAGE analysis of purified mutant endonuclease V proteins. Proteins were stained with Coomassie Brilliant Blue R-250. Lane 1, E23H; lane 2, E23C; lane 3, E23Q; lane 4, E23D; lane 5, wild type; lane 6, molecular weight markers. C, Western blot analysis of purified wild type and mutant endonuclease V proteins. FPLC (Mono S)-purified proteins were separated on a 15% SDS-PAGE gel, followed by Western blot analysis. The proteins were detected with mouse anti-endonuclease V polyclonal antibodies. The position of the prestained molecular weight markers are indicated in the margin. Lane 1, E23H; lane 2, E23C; lane 3, E23Q; lane 4, E23D; lane 5, wild type.



Purification of the Mutant Endonuclease V Proteins

The mutant enzymes were purified and their concentration was determined as described under ``Experimental Procedures.'' The concentrations obtained ranged from 50 to 100 µg/ml, and the yield was between 200 and 400 µg of mutant protein from each 2-liter culture. All four mutant endonuclease V proteins were identified by electrophoresis on a 15% SDS-PAGE gel followed by staining with Coomassie Brilliant Blue R-250. There were no discernable contaminants, and each of the mutant proteins appeared as a single discrete band (Fig. 2B). The purified proteins were also subjected to immunoblot analysis (Fig. 2C). Each preparation was found to be free of nonspecific DNA nicking activity on supercoiled pBR322 DNA (data not shown).

Amino-terminal Sequencing of Mutant Endonuclease V Proteins

Mutations at Glu-23 of endonuclease V were also confirmed by amino-terminal sequencing. A total of 25 cycles were run in order to observe the amino acid change at position 23. Consistent with previous results using the wild type enzyme in which the amino-terminal methionine had been removed, all of the mutant proteins had their amino-terminal methionine post-translationally cleaved in E. coli. The amino acid sequence analyses revealed that the desired changes (E23H, E23C, E23Q, and E23D) were accomplished in all four mutant proteins (Table 3). In the case of the E23C mutant, the cysteine residue at position 23 was not detected due to the hydrolysis of the unprotected cysteine side chain.



To assess the survival level after UV irradiation for each of the mutants, E. coli AB2480 (uvrA, recA), which had been transformed with plasmids containing the mutant den V genes, were irradiated with UV light for increasing periods of time. Evidence for the in vivo activity of the mutant enzymes can be assessed by measuring the colony-forming ability of these cells following UV exposure, since even one unrepaired pyrimidine dimer can be lethal to the cell(21) . Cells containing the wild type den V gene exhibited enhanced UV survival, compared to the survival detected in cells containing the mutant den V gene (Fig. 3). The survival of the E23C, E23Q, and E23D mutants was comparable to the levels seen in the parental strain containing only pGX2608 (den V). However, E23H displayed a better survival, although it was much lower than that observed in the wild type.


Figure 3: Determination of colony forming ability following UV-irradiation. E. coli AB2480 (uvrA, recA) harboring pGX2608 (den V), pGX2608-den V, or the pGX2608-den V mutant constructs (E23H, E23C, E23Q, E23D) were grown to confluence at 30 °C, diluted in growth medium (LB containing 100 µg/ml ampicillin), and spread onto plates containing LB agar plus ampicillin. Plates were UV-irradiated at 3.0 microwatts/cm^2 for increasing times and incubated at 30 °C for 36 h in the dark. Survival was measured as a function of colony-forming ability. , AB2480 pGX2608-den V; Delta, AB2480 pGX2608-den V; box, AB2480 pGX2608-den V His-23; circle, AB2480 pGX2608-den V Cys-23; , AB2480 pGX2608-den V Gln-23; bullet, AB2480 pGX2608-den V Asp-23.



Pyrimidine Dimer-specific Nicking Activity on Plasmid DNA

The pyrimidine dimer-specific nicking activity of the pure Glu-23 mutant proteins was evaluated using different experimental conditions. Form I pBR322 was UV-irradiated to produce approximately 25 dimers per pBR322 DNA molecule(22) . In one assay, UV-irradiated form I DNA was incubated with increasing concentrations of the wild type or mutant enzymes for 30 min at 37 °C. Wild type endonuclease V is known to recognize the pyrimidine dimer containing sites and make single-strand nicks that can convert form I (supercoiled) to form II (nicked circular) DNA. Relative enzyme activity was determined as a function of the loss of form I DNA. This assay was performed at different pH values, taking into consideration the E23H and E23C mutants. The imidazole side chain of histidine is unique, in that it has a pK(a) near neutrality and can therefore gain or lose protons by small changes in the local cellular environment. The pK(a) of cysteine could also be depressed within the range of pH in this assay. In the reactions performed at pH 6.0, the wild type enzyme showed considerable loss of form I DNA with 1-100 ng of enzyme (Fig. 4A). The only mutant which catalyzed any loss of form I DNA was the E23D mutant. However, the percentage loss of form I DNA in this mutant was much lower than the wild type. The other three mutants (E23H, E23C, and E23Q) were completely deficient in incision, which indicated that these enzymes are catalytically inactive on cyclobutane dimers. The time course nicking assay also revealed a small loss of form I DNA catalyzed by the E23D mutant; however, it took 100 times more of the E23D protein as compared to the wild type to effect the same decrease in form I DNA (Fig. 4B). Similar experiments were carried out at pH 6.8 (Fig. 4C) and pH 8.2 (Fig. 4D), and the trend seen in the loss of form I DNA among the mutant and wild type endonuclease V proteins was similar to that observed in reactions performed at pH 6.0.


Figure 4: Pyrimidine dimer-specific nicking activity of endonuclease V mutants. Wild type or mutant endonuclease V was added in varying concentrations to UV-irradiated form I pBR322 plasmid DNA. Following incubation at 37 °C for 30 min, the remaining percent form I DNA was determined following separation of DNA by agarose gel electrophoresis. A, enzyme titration at pH 6.0. B, time course assay at pH 6.8: wild type (1 ng) or mutant endonuclease V (100 ng) was added to UV-irradiated form I pBR322 plasmid DNA. Following incubation at 37 °C for 0 to 120 min, the remaining percent form I DNA was determined after agarose gel electrophoresis. Inset, comparison of in vitro dimer incision reaction catalyzed by wild type and E23D mutant endonuclease V in UV-irradiated form I DNA. Note that 100 times more E23D mutant enzyme is used when compared to wild type. C, enzyme titration at pH 6.8. D, enzyme titration at pH 8.2. box, wild type; bullet, E23H; , E23C; , E23Q; Delta, E23D.



Thymine Dimer-specific Nicking Activity on Synthetic Oligonucleotides

To further assess the ability of the mutant endonuclease V proteins to nick pyrimidine dimer-containing DNA, a highly sensitive in vitro assay was employed. A 49-base oligonucleotide containing a cis-syn cyclobutane thymine dimer, annealed to its complementary strand, was used as the substrate in this assay. The ability of the wild type and the various mutant endonuclease V proteins to recognize and nick the thymine dimer-containing DNA was monitored. The enzyme can generate the cleavage product only if it can recognize the thymine dimer and catalyze both the DNA glycosylase and AP lyase reactions. However, if there is only DNA glycosylase activity, treatment of the reaction products with piperidine results in cleavage of the phosphodiester bond at residual AP sites. Four different concentrations of either wild type or mutant enzyme were used in this assay (Fig. 5, A and B). One nanogram of the wild type endonuclease V was able to cleave all of the available substrate. The only mutant which exhibited extremely low levels of nicking activity (less than 1% of the wild type) was the E23D mutant, and this was achieved at a much higher concentration than wild type enzyme. Treating the samples with piperidine after the enzyme reaction did not significantly increase the amount of cleavage products either in the wild type or in the E23D mutant reactions. However, piperidine treatment resulted in -elimination, where the C-O-P bond 5` to the AP site is broken (releasing the deoxyribose sugar moiety), leaving a 3`-phosphate end. As expected, the -elimination reaction products migrated with a faster mobility compared to the beta-elimination products.


Figure 5: Thymine dimer-specific nicking activity on a synthetic DNA containing a site-specific thymine dimer. An oligonucleotide containing a cis-syn thymine dimer (CS 49-mer) was reacted with E23D mutant or wild type protein in duplicate for 30 min at 37 °C. One set of reaction mixtures were treated with piperidine for 30 min at 90 °C. The reaction products were separated by electrophoresis on a 15% polyacrylamide gel. A, lanes 1-5, E23D at 0, 0.1, 1, 10, and 100 ng; lanes 6-10, E23D at 0, 0.1, 1, 10, and 100 ng, followed by piperidine treatment. B, lanes 1-5, wild type at 0, 0.1, 1, 10, and 100 ng; lanes 6-10, wild type at 0, 0.1, 1, 10, and 100 ng, followed by piperidine treatment.



AP Site-specific Nicking Activity on Synthetic Oligonucleotides

To assess the ability of the four mutants to incise DNA at AP sites, an oligonucleotide containing a site-specific AP site was prepared. A 49-base oligonucleotide containing a site-specific uracil was annealed to its complementary strand and reacted with uracil DNA glycosylase to obtain double-stranded DNA with a site-specific abasic site. The wild type and the mutant enzymes were reacted with this substrate. The wild type and the E23D mutant were able to cleave the phosphodiester bond at the AP site (Fig. 6). The amount of AP site-specific nicking by the E23D mutant was approximately 60% that of wild type. However, the E23D mutant seemed to have retained significantly more of the AP lyase activity when compared to its glycosylase activity, as demonstrated in the plasmid and thymine dimer-containing oligonucleotide nicking assays. There was a negligible amount of AP site-specific nicking by the E23Q mutant and no detectable nicking by either the E23H or E23C mutants (data not shown).


Figure 6: AP site-specific nicking activity using a synthetic oligonucleotide containing an abasic site. An oligonucleotide containing a site-specific abasic site (AP 49-mer) was reacted with E23D mutant or wild type protein for 30 min at 37 °C, and the reaction products were separated by electrophoresis on a 15% denaturing polyacrylamide gel. Oligonucleotide markers are indicated in the margin. Lanes 1-4, E23D at 0, 1, 10, and 100 ng; lanes 5-8, wild type at 0, 1, 10, and 100 ng; lane 9, AP 49-mer reacted with piperidine only.



Binding of Mutant Proteins to a cis-syn Dimer-containing Oligonucleotide

To ensure that the decrease in nicking activity of the four mutants was not due to a lack of binding to pyrimidine dimers, a gel shift assay was performed. Each of the four mutant proteins was incubated with a thymine dimer-containing 49-mer, which had been prepared as described above. All of the mutants successfully bound to the dimer-containing DNA (Fig. 7A), forming two bands of slower mobility than the free DNA. Since wild type endonuclease V normally nicks this substrate, it is not possible to detect binding of the wild type enzyme by this method. However, the addition of NaBH(4) to this reaction reduces the imino intermediate formed between enzyme and DNA (11) and thus can be used to covalently trap the wild type enzyme to dimer-containing DNA. Fig. 7B shows the binding of both wild type and E23Q proteins to the adducted 49-mer in the presence of either NaBH(4) or NaCl. The NaBH(4) had no effect on the binding of the E23Q mutant, as would be expected if no imino intermediate were formed between enzyme and DNA (compare lanes 2 and 4 or 3 and 5 of Fig. 7B). The wild type enzyme, on the other hand, bound well to the substrate only when NaBH(4) was present (lanes 7-10). In the absence of NaBH(4), the wild type enzyme nicked the DNA and only minimally bound to the intact 49-mer (lane 10). We interpret these results to mean that E23Q mutant does not form the imino intermediate with dimer containing DNA.


Figure 7: Binding of mutant endonuclease V protein to a pyrimidine dimer-containing oligonucleotide. A, a 49-base oligonucleotide with a site-specific cis-syn cyclobutane thymine dimer (CS 49-mer) was 5`-end-labeled with [-P]ATP, annealed to its complementary strand, and allowed to bind with each mutant protein for 15 min at 20 °C. Samples were subjected to electrophoresis through a nondenaturing polyacrylamide gel. The control lane C did not contain any protein. B, the substrate described in A was allowed to bind with different concentrations of the E23Q mutant or wild type in 100 mM NaCl or NaBH(4) as indicated. Binding reactions were for 15 min at 20 °C. Reaction products were separated as described in A. Lane 1, control with DNA only; lanes 2 and 4, 2.7 µg/ml E23Q; lanes 3 and 5, 6.7 µg/ml E23Q; lane 6, 0.26 µg/ml wild type; lanes 7 and 9, 2.6 µg/ml wild type; lanes 8 and 10, 26 µg/ml wild type.



Molecular Weight Determination of E23Q Bound to Thymine Dimer and Tetrahydrofuran-containing Oligonucleotides

To determine the molecular weight of each of the two bound complexes formed by the mutant proteins interacting with the dimer-containing 49-mer, complexes between E23Q and the 49-mer were separated by electrophoresis through native gels of different polyacrylamide concentrations. The resulting relative mobilities of the complexes and native protein molecular weight standards were then analyzed by an adaptation of Ferguson analysis^2(18, 19) . The results of these analyses are displayed in Fig. 8A. Subtracting the molecular weight determined for the DNA alone from the first bound complex results in a molecular weight of 16,000 for the enzyme as bound to the DNA in the faster mobility complex. The slower mobility complex was found to contain an enzyme component of 31 kDa. Thus, the first (faster mobility) complex is the result of one molecule of the E23Q mutant bound to the 49-mer, and the second complex (slower mobility) reflects two enzyme molecules bound to the DNA. Interestingly, wild type endonuclease V covalently trapped to the thymine dimer-containing DNA by NaBH(4) forms a single complexed band of the same mobility as the faster mobility E23QbulletDNA complex (16-kDa protein component), with no evidence for a slower mobility complex (Fig. 7B).


Figure 8: Determination of the molecular weights of E23Q mutant endonuclease V bound to an oligonucleotide-containing a site-specific cis-syn thymine dimer or a tetrahydrofuran residue. A, the E23Q mutant was bound to the CS 49-mer and subjected to nondenaturing polyacrylamide gel electrophoresis. The logarithm of the relative mobility (R) of the native protein molecular weight standards were plotted against polyacrylamide concentration and the resulting slopes were plotted versus the molecular weights of the standard proteins. circle, molecular weight markers; bullet, CS 49-mer alone; , CS 49-mer-E23Q lower molecular weight complex; , CS 49-mer-E23Q higher molecular weight complex. B, the E23Q mutant was bound to the THF 30-mer and subjected to polyacrylamide gel analysis as in A: circle, molecular weight standards; bullet, THF 30-mer alone; , THF 30-mer-E23Q lower molecular weight complex; , THF 30-mer-E23Q higher molecular weight complex.



The ability of the E23Q mutant to bind an oligonucleotide containing a site-specific tetrahydrofuran (THF) residue was also evaluated. A THF residue is an abasic site analog that is uncleavable by AP lyase enzymes. Wild type endonuclease V binds to a THF-containing 30-mer as a monomer, as determined by Ferguson analysis. (^3)This substrate was used in similar experiments with the E23Q mutant. The E23Q mutant bound to a THF-containing 30-mer to form two distinct complexes, which was similar to its behavior on the thymine dimer-containing 49-mer (data not shown). Upon evaluation of the molecular weight of the protein components within the two complexes, the faster mobility complex contained protein with an approximate molecular weight of 21,000, and the slower complex contained a protein component of 36 kDa (Fig. 8B). Again, these values approximately correspond to the molecular weight of one and two molecules of E23Q enzyme per DNA molecule, respectively.


DISCUSSION

Reductive methylation and site-directed mutagenesis studies of the NH(2) terminus of T4 endonuclease V have demonstrated that the alpha-amino group of the amino-terminal amino acid of the enzyme is directly involved in the chemical mechanism of the pyrimidine dimer-specific glycosylase and AP lyase activities(9, 10, 11) . The x-ray crystal structure of T4 endonuclease V has revealed the close proximity of Glu-23 to the primary alpha-amino group in the active site of the enzyme(1) , and site-directed mutagenesis of Glu-23 suggested that it might be a second catalytically important residue(13, 14) . However, there has been limited information regarding the specific role of Glu-23 in the catalytic mechanism.

Structural modifications in the enzyme, as a result of mutating a critical amino acid residue, could play a major role in altering the stability of the enzyme in the reaction pathway, as there could be conformational changes in the enzyme. There are at least two sets of data that bear on this subject. First, proteins which do not fold properly are rapidly degraded within E. coli. In this study, the accumulation of mutant endonuclease V molecules within cells was found to be on comparable levels with the wild type (Fig. 2A). This result indicates that the structural integrity of the mutant proteins is conserved, and that they fold as efficiently as the wild type protein. Second, each of the mutant proteins was able to specifically bind cyclobutane pyrimidine dimer-containing DNA (Fig. 7A). These data also strongly suggest that the protein has properly folded and that the mutants are only catalytically compromised.

As the intracellular accumulation of the mutant enzymes was normal, studies on the in vivo activity of the mutant enzymes were initiated. The UV survival of cells expressing the E23C, E23Q, and E23D mutant enzymes was indistinguishable from that observed in cells which did not contain the den V gene. However, the phenotypic expression of the His mutant showed enhanced UV survival compared to the parental strain containing the pGX2608 vector alone (Fig. 3). These results suggest that glutamic acid in position 23 is critical for the enzyme to perform its various activities, which culminate in the removal of UV-induced pyrimidine dimers. The increased survival level observed in the E23H mutant is rather intriguing, as it is contrary to the results obtained from in vitro studies which are discussed later. It is possible that, under in vivo conditions, the change E23H could yield an enzyme with a minimal amount of activity. Yet another hypothesis based on the results obtained with the E23H mutant is that the enzyme changed its substrate specificity to dipyrimidine dimers (6-4 photoproducts) which could explain the increased UV survival. This hypothesis is yet to be tested.

Having established by in vivo studies that mutations at Glu-23 do not enhance the UV survival of repair-deficient E. coli cells compared to those cells expressing wild type endonuclease V, it was important to assess the catalytic properties of the mutant enzymes under in vitro conditions. Towards this end, a variety of assays were performed using plasmid DNA and synthetic oligonucleotides as substrates with the results being that E23D retained detectable but less than 1% dimer-specific nicking activity but near wild type AP lyase activity, while none of the other mutants retained any catalytic activity.

As part of the proposed catalytic mechanism of endonuclease V, it is possible that the enzyme dissociates from the pyrimidine dimer substrate after the glycosylic bond has been cleaved, leaving behind an abasic site (Fig. 1). If the Glu-23 mutants were able to successfully cleave the glycosylic bond and dissociate from the pyrimidine dimer without concomitant cleavage of the phosphodiester bond by beta-elimination, the enzyme would exhibit higher levels of glycosylase activity relative to dimer-specific nicking activity. To evaluate the pyrimidine dimer-specific glycosylase activity of the Glu-23 mutants, the reaction products were treated with alkali to cleave the phosphodiester bond at residual AP sites. None of the mutants possessed higher levels of glycosylase activity when compared to dimer-specific nicking activity (data not shown).

The low levels of pyrimidine dimer-specific nicking activity seen in plasmid nicking assays by the E23D mutant are somewhat contradictory to the results obtained in previous studies, where it was shown that E23Q and E23D were completely devoid in glycosylase activity(13) . This necessitated further studies on the catalytic activities of the enzyme using synthetic oligonucleotides as the substrate. These are well defined substrates with a cis-syn thymine dimer, and these assays are highly sensitive when compared to the plasmid nicking assays. The assays with the oligonucleotide substrate containing the cis-syn thymine dimer demonstrated the complete loss of pyrimidine dimer-specific nicking activity in all the mutants except the E23D mutant, which retained <1% activity. The nicking assays with piperidine treatment showed that there was no glycosylase activity evident in the E23H, E23C, or E23Q mutants.

Wild type endonuclease V recognizes and incises AP sites to produce 3` alpha,beta-unsaturated aldehyde and 5`-phosphate termini(12) . An oligonucleotide (49-mer) containing a site-specific uracil was reacted with uracil DNA glycosylase to create a single AP site. The removal of uracil was verified by reacting the substrate with piperidine. The wild type and the mutant proteins were reacted with the 49-mer containing the site-specific AP site. The results demonstrated that E23D retained substantial (approximately 60%) AP lyase activity. Previous studies indicated that the E23D mutant had up to 20% AP lyase activity(14) . All the other replacements done in this study (E23H, E23C, and E23Q) resulted in the complete loss of AP lyase activity.

The proposed chemical mechanism of endonuclease V (Fig. 1) describes the formation of a covalent (imino) enzyme-substrate intermediate between the amino-terminal alpha-amino group and the C1` of the 5`-deoxyribose of the pyrimidine dimer substrate. This imino intermediate can be formed at an abasic site or at a dimer site concomitant with the glycosylase reaction. Stabilization of the charge of this imino intermediate could be accomplished through an acidic residue at the active site. It is possible that the carboxyl group in the side chain of Glu-23 stabilizes this protonated Schiff base intermediate. The alternative roles of Glu-23 could be to increase the nucleophilicity of the alpha-amino terminus or to participate in the protonation of the pyrimidine ring or the deoxyribose ring oxygen, thereby labilizing the N-glycosylic bond. Confirmation with in vitro studies that none of the mutant enzymes, including the conservative replacement E23D, possessed any significant glycosylase activity indicates that the presence of the carboxyl group is critical, and that the relative position of the carboxyl group of Glu-23 to the substrate is critical for glycosylase activity. The absence of abasic lyase activity in the other mutants (E23H, E23C, and E23Q) indicates that the carboxylate anion at position 23 plays a major role in the beta-elimination reaction. It has been suggested that Glu-23 could act as a general base in the abstraction of the pro-S 2`-hydrogen(12) . However, abstraction of the pro-S 2`-hydrogen can only be an additional role for E23, as it is very clear that Glu-23 is required for the glycosylase activity. It is also likely that aspartic acid at position 23 is not as efficient as the glutamic acid in the abstraction of this hydrogen, perhaps due to positional effects which would explain the reduced levels of AP lyase activity observed in the E23D mutant.

Ferguson analysis of the electrophoretic mobility shift assays with the E23Q mutant bound to both thymine dimer- and tetrahydrofuran-containing DNA demonstrated that both one and two molecules of the E23Q mutant could bind to these substrates. These results were in contrast to the findings with the wild type enzyme. The wild type enzyme, when covalently trapped to the CS 49-mer with NaBH(4), and, when noncovalently bound to the THF 30-mer, is consistently a monomer. There are two possible reasons for this difference. First, in the E23Q mutant, an important catalytic residue proposed to be in contact with DNA has been neutralized. This neutralization could increase the DNA binding affinity of the E23Q mutant over that of the wild type. Alternately, it has been hypothesized that endonuclease V self-associates to processively search DNA. Since wild type enzyme is trapped to dimer-containing DNA as a monomer, the enzyme may undergo a conformational change upon catalysis that dissociates the multimer. In this scenario, the E23Q mutant would not form the imino intermediate, and undergo this change; thus, the protein multimer would stay associated. Further experiments would be necessary to prove this hypothesis.

The site-specific alterations which were constructed and evaluated in this study have provided several insights into the reaction mechanism of the enzyme. They have shown that the presence of a carboxylate-containing side chain at position 23 is obligatory for glycosylase and AP lyase activity. Shortening the side chain of amino acid 23 by one carbon-carbon bond (E23D) has resulted in the loss of glycosylase activity and reduced levels of AP lyase activity. This indicates that there is considerable tolerance in the volume of the side chain at position 23 for the AP lyase activity to occur. The results presented in this work, combined with the observations from previous work, unequivocally demonstrate that Glu-23 is a critical residue in the catalytic function of T4 endonuclease V. The role of the alpha-amino moiety of the enzyme, which is involved in the formation of the covalent imino intermediate, is taken into consideration to explain the reaction mechanism. The results obtained from this work have facilitated a more informed discussion of the different biochemical roles of Glu-23 in the reaction mech-anism of endonuclease V and make the catalytically active site of the enzyme better understood.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants ES04091 and ACS FRA381. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of the American Cancer Society Research Award FRA-381. To whom correspondence and reprint requests should be addressed. Tel.: 409-772-2179; Fax: 409-772-1790; rslloyd{at}scms.utmb.edu.

(^1)
The abbreviations used are: AP, apurinic or apyrimidinic; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylamino)propanesulfonic acid; RF, replicative form; THF, tetrahydrofuran; FPLC, fast protein liquid chromatography.

(^2)
Sigma Technical Bulletin MKR-137.

(^3)
K. A. Latham, S. Rajendran, J. R. Carmical, J. C. Lee, and R. S. Lloyd, submitted for publication.


ACKNOWLEDGEMENTS

We thank Drs. J. S. Taylor and C. Smith (Washington University, St. Louis) for their generous gift of synthetic oligonucleotides containing a site-specific cis-syn thymine dimer. Thanks to Drs. C. Iden, F. Johnson, and A. Grollman (SUNY, Stony Brook) for providing oligonucleotides containing a synthetically produced tetrahydrofuran residue. Synthesis of oligonucleotides for site-directed mutagenesis and amino acid sequence analysis were performed by the Recombinant DNA Laboratory and the Protein Chemistry Core Facility, respectively, at the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston.


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