©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
T4 Endonuclease V Protects the DNA Strand Opposite a Thymine Dimer from Cleavage by the Footprinting Reagents DNase I and 1,10-Phenanthroline-Copper (*)

(Received for publication, August 30, 1994; and in revised form, December 18, 1994 )

Katherine Atkins Latham (1) (2)(§) John-Stephen Taylor (3) R. Stephen Lloyd (2)(¶)

From the  (1)Department of Biochemistry and the Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37232, the (2)Sealy Center for Molecular Science, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1071, and the (3)Department of Chemistry, Washington University, St. Louis, Missouri 63130-4899

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha-NH(2) 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.


INTRODUCTION

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 beta-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 beta-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 alpha-helical. The N terminus lies wedged between two of the three alpha-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.


EXPERIMENTAL PROCEDURES

Materials

Endonuclease V was purified from Escherichia coli AB2480 (recA, uvrA) cells transformed with a denV-containing expression vector as described previously (27) . T4 polynucleotide kinase (10,000 units/ml) was purchased from New England Biolabs Inc., and terminal deoxynucleotidyltransferase was from Pharmacia Biotech Inc. 3-Mercaptopropionic acid, piperidine, 1,10-phenanthroline, and 2,9dimethyl-1,10-phenanthroline were obtained from Sigma, and dimethyl sulfate was purchased from Eastman Kodak Co.

Preparation of the DNA Substrate

A 49-base pair oligodeoxynucleotide containing a site-specific cis,syn-cyclobutane thymine dimer (CS 49-mer) (^1)with the sequence 5`-AGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT-3` (where the underlined bases represent the position of the thymine dimer (28) ) was used in its double-stranded form for all footprinting experiments. This oligonucleotide, or its complementary sequence, was P-labeled at the 5` terminus using polynucleotide kinase and [-P]ATP. Labeling of the 3`-end of the dimer-containing strand was accomplished by using terminal deoxynucleotidyltransferase and dideoxy-[alpha-P]ATP.

DNase I Footprinting Reactions

Footprinting with DNase I was carried out as described previously (29, 30) with minor modifications. Labeled DNA (0.048 pmol), in a total volume of 20 µl, was incubated with or without 1.9 pmol of endonuclease V in buffer (25 mM sodium phosphate (pH 6.8), 1 mM EDTA, 100 mM KCl) and 20 mM freshly diluted NaBH(4) for 90 min at 37 °C. The NaBH(4) was present to covalently trap the enzyme to the DNA. 180 µl of DNase I assay buffer (25 mM sodium phosphate (pH 6.8), 100 mM KCl, 1 mM CaCl(2), 2 mM MgCl(2), 100 µg/ml bovine serum albumin, 2 µg/ml poly(dIbulletdC)bulletpoly(dIbulletdC)) was then added, and the samples were warmed to 37 °C. Reactions were initiated by the addition of 5 µl of 0.005-0.01 mg/ml DNase I in dilution buffer (25 mM sodium phosphate (pH 6.8), 100 mM KCl, 1 mM CaCl(2), 2 mM MgCl(2)). After 1 min at 37 °C, reactions were terminated with 700 µl of DNase I stop buffer (645 µl of 100% ethanol, 5 µl of 1 mg/ml yeast tRNA, 50 µl of saturated ammonium acetate), and the DNA was precipitated at -70 °C. After washing with 70% ethanol, the DNA was dried, resuspended in loading buffer (95% (v/v) formamide, 20 mM EDTA, 0.025% (w/v) bromphenol blue, 0.025% (w/v) xylene cyanol), and analyzed by electrophoresis through a 15% polyacrylamide sequencing gel containing 8 M urea. Electrophoresis markers were produced for all footprinting experiments by reacting the labeled DNA (3.9 ng) with dimethyl sulfate (G reaction) or formic acid (G + A reaction) and treating with piperidine to cleave the DNA at the modified bases(31) .

1,10-Phenanthroline-Copper Footprinting

Copper complexed with 1,10-phenanthroline was used as a second footprinting reagent (32, 33, 34) . The double-stranded CS 49-mer (0.48 pmol), labeled on the 5`-end of either the dimer-containing strand or the complementary strand, was allowed to react with 240 pmol of endonuclease V (total reaction volume of 200 µl) in the presence of 100 mM NaBH(4) in reaction buffer (25 mM sodium phosphate (pH 6.8), 1 mM EDTA, 100 mM KCl). The NaBH(4) and the excess free enzyme were then removed from the oligonucleotide complexes or control DNA (no endonuclease V) by applying each sample to a 2-ml Sephadex G-25 column. The DNA was eluted with reaction buffer, and the radioactive fractions were used for subsequent reactions. A solution containing 0.45 mM copper sulfate and 2 mM 1,10-phenanthroline (4 µl) was added to 40 µl of the endonuclease V-bound or control CS 49-mers. 57 mM mercaptopropionic acid (4 µl) was used to initiate the 1,10-phenanthroline-copper-mediated cleavage, and the reactions were allowed to proceed for 30 s at 20 °C. Cleavage was terminated by the addition of 4 µl of 28 mM 2,9-dimethyl-1,10-phenanthroline. The cleaved or intact DNA was then ethanol-precipitated using poly(dIbulletdC)bulletpoly(dIbulletdC) as a carrier, dried, and resuspended in 10 µl of loading buffer. A fraction of each sample was subjected to scintillation counting to ensure that equivalent amounts of DNA were loaded into each well of the gel. The samples were separated by electrophoresis as described for the DNase I samples.

Methylation Protection Experiments

Appropriately labeled CS 49-mer DNA (0.096 pmol) was reacted with 0, 62, or 120 pmol of endonuclease V in 200 mM sodium cacodylate containing 1 mM EDTA and 100 mM NaBH(4). The covalently bound or free DNA was then subjected to methylation as described previously(31) . Briefly, 1 µl of concentrated dimethyl sulfate (DMS) was added and allowed to react with the DNA for 7 min at 20 °C. Reactions were stopped with 50 µl of DMS stop buffer (1.5 M sodium acetate (pH 7.0), 1 M 2-mercaptoethanol, 25 µg/ml poly(dIbulletdC)bulletpoly(dIbulletdC)), and the DNA was ethanol-precipitated twice. After a final ethanol wash, the DNA was dried, resuspended in 1 M freshly diluted piperidine, and heated to 90 °C for 30 min. The piperidine was removed under vacuum, and the DNA was washed twice with H(2)O. The dried DNA was then resuspended in loading buffer and analyzed by gel electrophoresis as described above.


RESULTS

Determination of the DNase I Footprint of Endonuclease V

DNase I has been widely used to locate specific protein-binding sites on DNA(30) . Specifically, one can see ``footprints'' of proteins on DNA where cleavage by DNase I is inhibited by the presence of a protein bound to the DNA. The interaction of endonuclease V with a 49-base pair oligonucleotide containing a site-specific cis,syn-cyclobutane thymine dimer was investigated using DNase I. The CS 49-mer was P-labeled on either the 5`- or 3`-end of the dimer-containing strand or on the 5`-end of the complementary strand. Endonuclease V was then covalently trapped to this CS 49-mer by reaction in the presence of NaBH(4). NaBH(4) has been previously shown to reduce the imino intermediate between the enzyme and DNA to form a stable covalent complex(17) . Labels were required on both ends of the dimer-containing strand because the enzyme was trapped to the DNA at the thymine dimer site, preventing analysis of the cleavage patterns 5` to the dimer on the 3`-labeled strand and 3` to the dimer on the 5`-labeled strand.

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(4) 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. (^2)


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.



Determination of the 1,10-Phenanthroline-Copper Complex Footprint

Because DNase I is an enzymatic nuclease and larger than endonuclease V (30.4 versus 16 kDa, respectively), the observed region of DNase I protection may overestimate the actual size of the DNA region involved in the interaction with endonuclease V. To further examine the contacts made on the CS 49-mer by endonuclease V, footprinting experiments were performed using a 1,10-phenanthroline-copper complex. This chemical nicks DNA in an oxygen-dependent manner, primarily via an oxidative attack on the C-1` hydrogen of the deoxyribose. The chemistry of the 1,10-phenanthroline-copper complex has been shown to be minor groove-specific(33, 34, 35) .

Endonuclease V was covalently trapped to the labeled CS 49-mer in the presence of NaBH(4) 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(4) 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(4), 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.



Methylation Protection Experiments to Probe Minor Versus Major Groove Specificity

Reaction of DNA with DMS has long been recognized as a tool to investigate the change in accessibility of the major versus minor groove of DNA due to binding of protein (36, 37) . DMS methylates N-7 of guanine (major groove) and N-3 of adenine (minor groove). Proteins that protect the DNA from DMS-mediated methylation of adenines are likely to obscure the minor groove upon DNA binding, whereas protection of guanine residues reflects protein contacts within the major groove. Hypersensitive sites are thought to reflect the generation of hydrophobic pockets in which the local concentration of DMS is higher than in solution.

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(4)(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(4) 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.




DISCUSSION

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(4), 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(4) 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(4). The enzyme may form a tighter complex with DNA before the glycosylase incision. NaBH(4) 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(4)-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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants ES04091 (to R. S. L.) and CA40463 (to J.-S. T.) and by American Cancer Society Award FRA-381 (to R. S. L.). 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.

§
Supported by a National Science Foundation predoctoral fellowship.

To whom correspondence should be addressed. Tel.: 409-772-2179; Fax: 409-772-1790.

(^1)
The abbreviations used are: CS 49-mer, cis,syn-cyclobutane dimer-adducted 49-base oligodeoxynucleotide; DMS, dimethyl sulfate.

(^2)
K. A. Latham, S. Rajendran, J. R. Carmical, J. C. Lee, and R. S. Lloyd, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank J. Russ Carmical and Melissa Prince for purification of endonuclease V and Colin A. Smith for synthesis of the CS 49-mer. We also thank M. L. Dodson for many useful discussions and Gary J. Latham for critical reading of this manuscript.


REFERENCES

  1. Lloyd, R. S., Hanawalt, P. C. & Dodson, M. L. (1980) Nucleic Acids Res. 8, 5113-5127 [Abstract]
  2. Ganesan, A. K., Seawell, P. C., Lewis, R. J. & Hanawalt, P. C. (1986) Biochemistry 25, 5751-5755 [Medline] [Order article via Infotrieve]
  3. Gruskin, E. A. & Lloyd, R. S. (1986) J. Biol. Chem. 261, 9607-9613 [Abstract/Free Full Text]
  4. Gruskin, E. A. & Lloyd, R. S. (1988) J. Biol. Chem. 263, 12728-12737 [Abstract/Free Full Text]
  5. Dowd, D. R. & Lloyd, R. S. (1989) J. Mol. Biol. 208, 701-707 [Medline] [Order article via Infotrieve]
  6. Dowd, D. R. & Lloyd, R. S. (1989) Biochemistry 28, 8699-8705 [Medline] [Order article via Infotrieve]
  7. Gordon, L. K. & Haseltine, W. A. (1980) J. Biol. Chem. 255, 12047-12050 [Abstract/Free Full Text]
  8. Seawell, P. C., Simon, T. J. & Ganesan, A. K. (1980) Biochemistry 19, 1685-1691 [Medline] [Order article via Infotrieve]
  9. McMillan, S., Edenberg, H. J., Radany, E. H., Friedberg, R. C. & Friedberg, E. C. (1981) J. Virol. 40, 211-223 [Medline] [Order article via Infotrieve]
  10. Weiss, B. & Grossman, L. (1987) Adv. Enzymol. Relat. Areas Mol. Biol. 60, 1-34 [Medline] [Order article via Infotrieve]
  11. Kim, J. & Linn, S. (1988) Nucleic Acids Res. 16, 1135-1141 [Abstract]
  12. Bailly, V., Sente, B. & Verly, W. G. (1989) Biochem. J. 259, 751-759 [Medline] [Order article via Infotrieve]
  13. Manoharan, M., Mazumder, A., Ransom, S. C. & Gerlt, J. A. (1988) J. Am. Chem. Soc. 110, 2690-2691
  14. Mazumder, A., Gerlt, J. A., Absalon, M. J., Stubbe, J. & Bolton, P. H. (1989) J. Am. Chem. Soc. 111, 8029-8030
  15. Mazumder, A., Gerlt, J. A., Absalon, M. J., Stubbe, J., Cunningham, R., Withka, J. & Bolton, P. H. (1991) Biochemistry 30, 1119-1126 [Medline] [Order article via Infotrieve]
  16. Schrock, R. D., III & Lloyd, R. S. (1991) J. Biol. Chem. 266, 17631-17639 [Abstract/Free Full Text]
  17. Dodson, M. L., Schrock, R. D., III & Lloyd, R. S. (1993) Biochemistry 32, 8284-8290 [Medline] [Order article via Infotrieve]
  18. Schrock, R. D., III & Lloyd, R. S. (1993) J. Biol. Chem. 268, 880-886 [Abstract/Free Full Text]
  19. Morikawa, K., Matsumoto, O., Tsujimoto, M., Katayanagi, K., Ariyoshi, M., Doi, T., Ikehara, M., Inaoka, T. & Ohtsuka., E. (1992) Science 256, 523-526 [Medline] [Order article via Infotrieve]
  20. Recinos, A., III & Lloyd, R. S. (1988) Biochemistry 27, 1832-1838 [Medline] [Order article via Infotrieve]
  21. Stump, D. G. & Lloyd, R. S. (1988) Biochemistry 27, 1839-1843 [Medline] [Order article via Infotrieve]
  22. Lee, B. J., Ohkubo, T., Ikehara, M., Doi, T., Morikawa, K., Kyogoku, Y., Osafune, T., Iwai, S. & Ohtsuka, E. (1994) Biochemistry 33, 57-64 [Medline] [Order article via Infotrieve]
  23. Kemmink, J., Boelens, R., Koning, T. M. G., Kaptein, R., van der Marel, G. A. & van Bloom, J. H. (1987) Eur. J. Biochem. 162, 37-43 [Abstract]
  24. Kemmink, J., Boelens, R., Koning, T., van der Marel, G. A., van Bloom, J. H. & Kaptein, R. (1987) Nucleic Acids Res. 15, 4645-4653 [Abstract]
  25. Taylor, J.-S., Garrett, D. S., Brockie, I. R., Svoboda, D. L. & Telser, J. (1990) Biochemistry 29, 8858-8866 [Medline] [Order article via Infotrieve]
  26. Iwai, S., Maeda, M., Shimada, Y., Hori, N., Murata, T., Morioka, H. & Ohtsuka, E. (1994) Biochemistry 33, 5581-5588 [Medline] [Order article via Infotrieve]
  27. Prince, M. A., Friedman, B., Gruskin, E. A., Schrock, R. D., III & Lloyd, R. S. (1991) J. Biol. Chem. 266, 10686-10693 [Abstract/Free Full Text]
  28. Smith, C. A. & Taylor, J.-S. (1993) J. Biol. Chem. 268, 11143-11151 [Abstract/Free Full Text]
  29. Brenowitz, M., Senear, D. F. & Kingston, R. E. (1989) Current Protocols in Molecular Biology , John Wiley and Sons, Boston, MA
  30. Galas, D. J. & Schmitz, A. (1978) Nucleic Acids Res. 5, 3157-3170 [Abstract]
  31. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560 [Medline] [Order article via Infotrieve]
  32. Kuwabara, M. D. & Sigman, D. S. (1987) Biochemistry 26, 7234-7238 [Medline] [Order article via Infotrieve]
  33. Sigman, D. S. (1986) Acc. Chem. Res. 19, 180-186
  34. Sigman, D. S., Kuwabara, M. D., Chen, C.-H. B. & Bruice, T. W. (1991) Methods Enzymol. 208, 414-433 [Medline] [Order article via Infotrieve]
  35. Marshall, L. E., Graham, D. R., Reich, K. A. & Sigman, D. S. (1981) Biochemistry 20, 244-250 [Medline] [Order article via Infotrieve]
  36. Johnsrud, L. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 5314-5318 [Abstract]
  37. Siebenlist, U. & Gilbert, W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 122-126 [Abstract]
  38. Augustine, M. L., Hamilton, R. W., Dodson, M. L. & Lloyd, R. S. (1991) Biochemistry 30, 8052-8059 [Medline] [Order article via Infotrieve]
  39. Hori, N., Doi, T., Karaki, Y., Kikuchi, M., Ikehara, M. & Ohtsuka, E. (1992) Nucleic Acids Res. 20, 4761-4764 [Abstract]
  40. Doi, T., Recktenwald, A., Karaki, Y., Kikuchi, M., Morikawa, K., Ikehara, M., Inaoka, T., Hori, N. & Ohtsuka, E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9420-9424 [Abstract]
  41. Lahm, A. & Suck, D. (1991) J. Mol. Biol. 221, 645-667
  42. Suck, D., Lahm, A. & Oefner, C. (1988) Nature 332, 464-468 [CrossRef][Medline] [Order article via Infotrieve]
  43. Weston, S. A., Lahm, A. & Suck, D. (1992) J. Mol. Biol. 226, 1237-1256 [Medline] [Order article via Infotrieve]
  44. Frederick, C. A., Grable, J., Melia, M., Samudzi, C., Jen-Jacobsen, L., Wang, B. C., Greene, P., Boyer, H. W. & Rosenberg, J. M. (1984) Nature 309, 327-331 [Medline] [Order article via Infotrieve]
  45. Kuwabara, M. D., Yoon, C., Goyne, T. E., Thederahn, T. B. & Sigman, D. S. (1986) Biochemistry 25, 7401-7408 [Medline] [Order article via Infotrieve]
  46. Baer, M. & Sancar, G. B. (1989) Mol. Cell. Biol. 9, 4777-4788 [Medline] [Order article via Infotrieve]
  47. Tchou, J., Michaels, M. L., Miller, J. H. & Grollman, A. P. (1993) J. Biol. Chem. 268, 26738-26744 [Abstract/Free Full Text]

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