(Received for publication, August 30, 1994; and in revised form, December 18, 1994 )
From the
The glycosylase/abasic lyase T4 endonuclease V initiates the
repair of ultraviolet light-induced pyrimidine dimers. This enzyme
forms an imino intermediate between its N-terminal -NH
group and C-1` of the 5`-residue within the dimer. Sodium
borohydride was used to covalently trap endonuclease V to a 49-base
pair oligodeoxynucleotide containing a site-specific cyclobutane
thymine dimer. The bound and free oligonucleotides were then subjected
to nuclease protection assays using DNase I and a complex of
1,10-phenanthroline- copper. There was a large region of protection
from both nucleases produced by endonuclease V evident on the strand
opposite and asymmetrically opposed to the dimer. Little protection was
seen on the dimer-containing strand. The existence of a footprint with
the 1,10-phenanthroline-copper cleavage agent indicated that
endonuclease V was interacting with the DNA predominantly via the minor
groove. Methylation by dimethyl sulfate yielded no areas of protection
when endonuclease V was covalently attached to the DNA, indicating that
the protein may closely approach the DNA without direct contact with
the bases near the thymine dimer. The Escherichia coli proteins Fpg and photolyase display a very different pattern of
nuclease protection on their respective substrates, implying that
endonuclease V recognizes pyrimidine dimers by a novel mechanism.
Endonuclease V, encoded by the denV gene of
bacteriophage T4, initiates DNA repair at the site of ultraviolet
light-induced pyrimidine dimers. The enzyme binds nontarget DNA in a
salt-dependent manner and searches along the DNA by facilitated
diffusion to locate its target
site(1, 2, 3, 4, 5, 6) .
Upon binding a pyrimidine dimer, the enzyme catalyzes a glycosylic bond
scission, removing the base of the 5`-pyrimidine within the
dimer(7, 8, 9) . The phosphodiester backbone
is then cleaved via a -elimination
mechanism(10, 11, 12, 13, 14, 15) .
The N-terminal amino group of endonuclease V has been found to
participate directly in the cleavage reaction by forming an imino
intermediate between the enzyme and C-1` of the 5`-sugar within the
dimer(16, 17, 18) . This imino intermediate
promotes
-elimination chemistry and can be reduced by sodium
borohydride, which covalently traps the enzyme to the DNA(18) .
The structure of endonuclease V has been determined by x-ray
crystallography(19) . The enzyme consists of a single domain
and is predominantly -helical. The N terminus lies wedged between
two of the three
-helices found in the enzyme. The C terminus,
which has been implicated in dimer-specific recognition by
site-directed mutagenesis studies (20, 21) and NMR
experiments(22) , lies in a loop far removed from the
N-terminal active site. The large separation of these two important
regions leads to questions of how the enzyme interacts with a
pyrimidine dimer site within DNA. The presence of a cis,syn-thymine dimer has been found by NMR
spectroscopy to only minimally affect the normal B-form DNA structure (22, 23, 24, 25) . It remains
unclear how endonuclease V recognizes this substrate since a structure
of endonuclease V complexed with pyrimidine dimer-containing DNA is not
yet available. Recent work with substrate analogs has indicated that
the enzyme interacts with dimer-containing DNA via the minor
groove(26) , although the specific protein-DNA contacts have
not been determined.
In this study, we have utilized three different footprinting techniques to further characterize the interaction of endonuclease V with pyrimidine dimer-containing DNA. Sodium borohydride was used to covalently trap the enzyme to a 49-base pair oligodeoxynucleotide containing a site-specific cis,syncyclobutane thymine dimer. The oligonucleotide-enzyme complex was then subjected to treatment with DNase I, a 1,10-phenanthroline-copper complex, or dimethyl sulfate in order to map the DNA contacts that endonuclease V makes when bound to a thymine dimer.
Reaction of DNase I with the CS 49-mer alone resulted in a ladder of cleavage products of various intensities (Fig. 1, lanes3 and 5), reflecting the inherent sequence dependence of DNase I. Covalently trapping endonuclease V to the DNA labeled on the dimer-containing strand produced a band with a very slow mobility (Fig. 1, A and B, lane2). DNase I digestion of these enzyme-DNA complexes resulted in distinct bands only below the dimer site, indicating that all fragments containing the dimer were complexed with the enzyme and thus retarded in mobility. Endonuclease V was found to protect the dimer-containing DNA strand only minimally from DNase I incision. There was protection evident 1 base 5` to the dimer site (Fig. 1A) and at a single base 4 residues removed from the dimer site on the 3`-side (Fig. 1B and 2), and these residues were consistently protected when the experiment was repeated.
Figure 1:
DNase I
protection assay of endonuclease V bound to the CS 49-mer. A 49-base
pair oligodeoxynucleotide containing a site-specific cis,syn-cyclobutane thymine dimer was P-labeled at the 5`-terminus (A) or the
3`-terminus (B) of the dimer-containing strand or at the
5`-terminus of the complementary strand (C). The appropriately
labeled substrates were reacted with endonuclease V in the presence of
20 mM NaBH
to covalently trap the enzyme to the
DNA, and the complexes were cleaved by DNase I as described under
``Experimental Procedures.'' G and G + A refer to the marker lanes in which the CS 49-mer was subjected to
Maxam-Gilbert G and G + A reactions(31) . Lane1, CS 49-mer alone, no DNase I; lane2,
CS 49-mer bound to endonuclease V; lane3, CS 49-mer
subjected to 0.1 (A) or 0.05 (B and C)
µg of DNase I; lane 4, CS 49-mer bound to endonuclease V
and subjected to 0.1 (A) or 0.05 (B and C)
µg of DNase I; lane 5, CS 49-mer subjected to 0.25 (A) or 0.025 (B and C) µg of DNase I; lane 6, CS 49-mer bound to endonuclease V subjected to 0.25 (A) or 0.025 (B and C) µg of DNase I.
The position of the thymine dimer is indicated (A, <; B and C, outlinedletters). Arrows indicate the regions of protection
observed.
When the complementary
strand was end-labeled and subjected to DNase I treatment with and
without bound endonuclease V, an area of 10 bases was found to be
protected from DNase I cleavage by the enzyme (Fig. 1C and Fig. 2). This region was asymmetrically situated
opposite the thymine dimer. Thus, endonuclease V predominantly
interacts with the DNA on the strand opposite the dimer and has little
contact with the dimer-containing strand. The asymmetrical position of
the protected region probably reflects the fact that endonuclease V
binds to a pyrimidine dimer as a monomer, as no dyad symmetry is
evident. Indeed, the enzyme has been shown to bind to both thymine
dimer-containing and reduced abasic sitecontaining DNAs as a monomer. ()
Figure 2: Schematic diagram of the residues protected by endonuclease V from DNase I cleavage. Arrows denote the DNA residues protected from cleavage by DNase I.
Endonuclease
V was covalently trapped to the labeled CS 49-mer in the presence of
NaBH as described. In the 1,10-phenanthroline-copper
footprinting method, the cupric ion is reduced by thiol
(mercaptopropionic acid) to a cuprous complex, allowing for the
generation of hydrogen peroxide in situ(33, 34, 35) . Since NaBH
would greatly interfere with this reaction, it was removed prior
to addition of the 1,10-phenanthroline-copper complex. The footprint
generated from the binding of endonuclease V to the CS 49-mer
5`-labeled on the dimer-containing strand yielded one region of
protection: the 3 residues immediately 5` to the dimer (Fig. 3A and 4). This result is interesting when one
considers that the DNase I footprint was actually smaller on this
strand. The footprint generated from the 3`-labeling of the
dimer-containing strand yielded no regions of obvious protection (data
not shown). Endonuclease V bound to the CS 49-mer labeled on the
complementary strand, as in the DNase I protection experiments, yielded
a sizable footprint (Fig. 3B and Fig. 4). Ten
residues, again asymmetrically opposed to the thymine dimer, were
protected from 1,10phenanthroline-copper-mediated cleavage by
endonuclease V. These protected residues were 2 bases removed to the
5`-side of the 10-base DNase I footprint. In other words, the footprint
on the strand opposite the dimer spanned 2 residues past the dimer site
on the 5`-side to 6 residues past the dimer on the 3`-side. The
presence of this region of protection indicated that endonuclease V was
binding to its target site via the minor groove since the
1,10-phenanthroline-copper complex is known to be specific for the
minor groove.
Figure 3:
1,10-Phenanthroline-copper cleavage
protection assay of endonuclease V bound to the CS 49-mer. The CS
49-mer was P-labeled at the 5`terminus of the
dimer-containing strand (A) or at the 5`-terminus of the
complementary strand (B). Endonuclease V was covalently
attached to the DNA substrates by reaction in the presence of
NaBH
, and the oligonucleotides were subjected to cleavage
by a 1,10-phenanthroline-copper complex as described under
``Experimental Procedures.'' Lane1, CS
49-mer only, no addition of 1,10-phenanthrolinecopper; lane 2,
CS 49-mer bound to endonuclease V, no 1,10-phenanthroline-copper; lane3, CS 49-mer treated with
1,10-phenanthroline-copper for 30 s; lane 4, CS 49-mer bound
to endonuclease V and treated with 1,10-phenanthroline-copper for 30
s.
Figure 4: Schematic diagram of the residues protected by endonuclease V from 1,10-phenanthroline-copper-mediated cleavage. Arrows represent the DNA residues protected by endonuclease V.
Endonuclease V was covalently bound to each of the labeled CS
49-mers, and the complexes, along with free DNA controls, were
subjected to methylation by DMS (Fig. 5). Since cleavage of the
complementary strand opposite the thymine dimer by both DNase I and a
complex of 1,10-phenanthroline-copper was blocked by endonuclease V, we
expected methylation protection to be apparent on the 2 adenines
directly opposite the dimer. In fact, endonuclease V has been
previously reported to protect the 2 adenines directly across from a
thymine dimer from methylation by DMS when the enzyme was reacted with
a thymine dimer-containing 30-base oligonucleotide in the absence of
NaBH(26) . In contrast to the expected results, no
methylation protection was evident for any of the DNA samples
covalently trapped to endonuclease V, even on the adenines directly
opposite the thymine dimer (Fig. 5C). The differences
in these data may be due to slight alterations in DNA structure or
enzyme-DNA contacts caused by covalently attaching endonuclease V to
the DNA. The only difference in the methylation protection experiments
noted between free and bound DNAs was the existence of an extra band at
the thymine dimer site (Fig. 5, A and B, lanes2 and 3), reflecting a small amount of
thymine dimer-specific nicking by endonuclease V.
Figure 5:
Methylation protection assay of
endonuclease bound to the CS 49-mer. The CS 49-mer was P-labeled at the 5`-terminus (A) or the
3`-terminus (B) of the dimer-containing strand or at the
5`-terminus of the complementary strand (C). The DNA was
reacted with two different concentrations of endonuclease V in the
presence of NaBH
to covalently attach the enzyme to the
DNA. The free or bound DNA complexes were then methylated with DMS and
cleaved by piperidine and heat. Lane1, free CS
49-mer methylated with DMS; lane 2, CS 49-mer reacted with 62
pmol of endonuclease V and then methylated with DMS; lane 3,
CS 49-mer reacted with 120 pmol of endonuclease V and then methylated
with DMS.
The subject of damage recognition by DNA repair proteins has been studied in a number of different enzyme models. One of the best models for understanding DNA glycosylases and glycosylase/abasic lyases is endonuclease V from bacteriophage T4. The extensive characterization of endonuclease V includes: (i) site-directed mutagenesis studies that have pinpointed numerous residues important for nontarget DNA binding (5, 6, 38) , substrate recognition(20, 21) , and catalysis(18, 39, 40) ; (ii) determination of the crystal structure(19) ; and (iii) chemical modification studies that have led to the proposal of a catalytic mechanism(16, 17) . What still remains a mystery, however, is how the enzyme recognizes its pyrimidine dimer substrate. The enzyme is known to search DNA by a salt-dependent facilitated diffusion mechanism to locate its target site(1, 2, 3, 4, 5, 6) , and the C terminus has been shown to be important for pyrimidine dimer-specific binding(20, 21, 22) . A recent report has demonstrated that endonuclease V binds to a thymine dimer-containing oligonucleotide via the minor groove(26) . To further elucidate the mechanism by which endonuclease V interacts with pyrimidine dimer-containing DNA, we have employed two different footprinting techniques along with methylation protection experiments to map the DNA contacts around a thymine dimer site made by endonuclease V.
The catalytic mechanism of endonuclease V has been
shown to involve an imino intermediate that can be reduced by
NaBH, resulting in a dead-end covalent
product(16, 17) . To prevent endonuclease V from
incising and dissociating from the dimer-containing oligonucleotide
substrate, as it would during the normal course of reaction, NaBH
was used to reduce the imino intermediate and covalently trap the
enzyme to the DNA. Bonding of the protein to the DNA necessitated the
radiolabeling of both ends of the thymine dimer-containing strand of
the double-stranded oligonucleotide used in the study since any DNA
fragments containing the dimer site would be covalently attached to the
protein and would be uninformative.
Reaction of the CS 49-mer-endonuclease V complex with DNase I evidenced a small area of protection on the damaged strand, including single residues 1 base 5` and 4 bases 3` to the thymine dimer, and a much larger 10-base region of protection on the complementary strand when compared with DNase I cleavage of the free CS 49-mer. The protection observed 4 bases removed from the dimer may reflect the spanning of endonuclease V across one of the grooves of the DNA, blocking the access of DNase I. The footprint on the complementary strand is offset from the thymine dimer ( Fig. 2and Fig. 4), indicating that the enzyme binds to a thymine dimer asymmetrically, largely from the 5`-side of the dimer, but on the opposite DNA strand. Footprinting using 1,10-phenanthroline-copper as the nuclease results in similar areas of protection that are offset from the DNase I footprints by 2 bases. This change in pattern is the result of the manner in which DNase I binds to DNA. DNase I has been crystallized in the presence of DNA (41, 42, 43) and shown to bind to DNA in the minor groove, making phosphate contacts across the groove: four on one strand and two on the other. Thus, the DNase I contacts made with the DNA are asymmetrically arranged around the site of incision, explaining the 2-base difference in the boundaries of protection between DNase I and 1,10-phenanthroline-copper.
Because DNase I is much larger than the 1,10-phenanthroline-copper complex, we expected a larger endonuclease V footprint using DNase I as the reagent as compared with 1,10-phenanthroline-copper. Examination of the data from the two different techniques demonstrates that the regions of protection are almost identical in size (although 2 bases offset from one another, as mentioned above). The 1,10-phenanthroline-copper complex reacts with C-1` and C-4` of deoxyribose, which are in the minor groove(33, 34) , making the reagent minor groove-specific. Indeed, EcoRI, shown by crystallography to interact with its cognate sequence via the major groove(44) , does not protect DNA from cleavage by 1,10-phenanthroline-copper(45) . The fact that endonuclease V protects the DNA from cleavage by 1,10-phenanthroline-copper indicates that either the enzyme contacts the DNA in the minor groove or the enzyme perturbs the minor groove such that the chemical nuclease can no longer bind efficiently.
The covalent attachment of endonuclease V
to thymine dimer-containing DNA did not affect the methylation of the
DNA by DMS. This result was surprising given the level of protection
from cleavage by DNase I and 1,10-phenanthroline-copper. We expected
the 2 adenines directly across from the thymine dimer to be protected
from DMS methylation by endonuclease V since adenines are methylated by
DMS on N-3, which lies in the minor groove. A recent report has shown
that reaction of a dimer-containing oligonucleotide with endonuclease V
(without trapping the enzyme to the DNA) resulted in methylation
protection of the 2 adenines across from the thymine
dimer(26) . When 2 guanines were mispaired opposite the thymine
dimer, no methylation protection was observed, indicating that
endonuclease V protects the DNA strand opposite the dimer from
methylation in the minor groove, but not from methylation in the major
groove. In the previous study(26) , methylation protection of
the dimer-containing strand was not investigated, as the enzyme nicked
the DNA in the course of the reaction. To ensure that our observed
absence of methylation protection was not due to methylation of the
enzyme causing a disruption of crucial enzyme-DNA contacts, a control
experiment was performed in which just the enzyme was treated with DMS
(data not shown). The DMS-treated endonuclease V displayed identical
thymine dimer-specific nicking activity compared with the untreated
enzyme, indicating that the DMS treatment conditions used in our
protection assay do not affect the binding affinity or catalytic
competence of the enzyme. The lack of methylation protection provided
by the covalently attached endonuclease V in our experiments may
reflect slight differences in the enzyme-DNA complex caused by
reduction of the imino intermediate by NaBH. The enzyme may
form a tighter complex with DNA before the glycosylase incision.
NaBH
reduces the imino intermediate formed after the
5`-thymine of the dimer has already been released, perhaps trapping the
enzyme in a slightly different conformation than before catalysis. The
conformation of the enzyme after reduction of the imino intermediate
may not contact the DNA as tightly, allowing a small molecule like DMS
to come in contact with the DNA, while still obscuring larger molecules
like DNase I or 1,10-phenanthroline-copper. One way to determine
whether or not NaBH
-mediated reduction of the enzyme-DNA
intermediate changes the overall footprint characteristics would be to
perform footprinting experiments with catalytically inactive mutants of
endonuclease V. For instance, the E23Q mutant has been shown to be
catalytically inactive, yet retains the capability to bind thymine
dimer-containing DNA(19, 40) . DNase I and
1,10-phenanthroline-copper protection assays could be performed using
this mutant, and the results compared with those from the covalently
trapped wild-type enzyme. Furthermore, the E23Q mutant and wild-type
proteins could be used in footprinting experiments on a
tetrahydrofuran-containing substrate, which is a noncleavable abasic
site analog, to determine whether or not the E23Q mutant retained the
same DNA contacts as the wild type.
The interaction of endonuclease V with its substrate DNA is remarkably different from some of the other DNA repair enzymes examined by footprinting techniques. We have shown that endonuclease V binds to the thymine dimer via the minor groove, making asymmetric DNA contacts primarily with the complementary strand. E. coli photolyase, which recognizes pyrimidine dimers and catalyzes a light-dependent photoreversal reaction, has been found to leave symmetrical methidiumpropyl-EDTA footprints of 6-7 bases on the pyrimidine dimer-containing strand and of 7-8 bases on the complementary DNA strand. Alkylation interference experiments have shown that photolyase makes contacts predominantly with the major groove, although a portion of the enzyme probably lies in the minor groove(46) . The Fpg protein, which is a glycosylase/abasic lyase that is specific for 8-oxo-dG and the ring-opened formamidopyrimidine adduct, has been shown to leave a 5-base footprint on a tetrahydrofuran-containing substrate when iron/EDTA was used as the nuclease. This region of protection was symmetrically located on the same strand as the damage, unlike the footprint of endonuclease V(47) . Thus, photolyase, which recognizes the same damaged substrate as endonuclease V, and Fpg, which recognizes a different substrate but which has an activity similar to that of endonuclease V, both possess quite different modes of substrate recognition compared with endonuclease V. It remains to be determined whether the Micrococcus luteus UV endonuclease, which has the same substrate specificity and catalytic function as endonuclease V, will bind to substrate DNA in a manner similar to that of endonuclease V. It is possible that the T4 enzyme has evolved a unique method of recognizing its substrate.