(Received for publication, August 17, 1994; and in revised form, November 28, 1994)
From the
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
-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.
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 ()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
-NH
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
-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) and subsequent isolation of the complex, and (c) demonstration that the amino-terminal
-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
-NH
terminus at C-1` on the 5` sugar in
the pyrimidine dimer (panel B). The phosphodiester bond is
cleaved by
-elimination facilitated by the removal of pro-S 2` hydrogen, resulting in an
,
-unsaturated aldehyde and
a 3`-phosphate as shown in panel D(12) . However, the
enzyme can also dissociate without undergoing the
-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
-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 (-NH
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
-NH
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.
Scheme I: Purification scheme of mutant endonuclease V proteins.
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.
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.
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
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
;
, AB2480
pGX2608-den V
;
, AB2480 pGX2608-den V
His-23;
, AB2480 pGX2608-den V
Cys-23;
, AB2480 pGX2608-den V
Gln-23;
, AB2480 pGX2608-den V
Asp-23.
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. , wild type;
,
E23H;
, E23C;
, E23Q;
,
E23D.
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.
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.
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
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.
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.
,
molecular weight markers;
, 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:
, molecular weight standards;
, 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. ()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.
Reductive methylation and site-directed mutagenesis studies
of the NH terminus of T4 endonuclease V have demonstrated
that the
-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
-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 -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` ,
-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 -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
-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
-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, 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 -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.