From the Laboratory of Structural Biology, National
Institute of Environmental Health Sciences, Research Triangle Park,
North Carolina 27709, ** Sealy Center for Molecular Science, University
of Texas Medical Branch, Galveston, Texas 77555, and ¶ Department
of Biochemistry, University of Connecticut Health Center,
Farmington, Connecticut 06032
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ABSTRACT |
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The amino-terminal 8-kDa domain of
DNA polymerase functions in binding single-stranded DNA (ssDNA),
recognition of a 5'-phosphate in gapped DNA structures, and as a
5'-deoxyribose phosphate (dRP) lyase. NMR and x-ray crystal structures
of this domain have suggested several residues that may interact with
ssDNA or play a role in the dRP lyase reaction. Nine of these residues
were altered by site-directed mutagenesis. Each mutant was expressed in
Escherichia coli, and the recombinant protein was purified
to near homogeneity. CD spectra of these mutant proteins indicated that
the alteration did not adversely affect the global protein structure.
Single-stranded DNA binding was probed by photochemical cross-linking
to oligo(dT)16. Several mutants (F25W, K35A, K60A, and
K68A) were impaired in ssDNA binding activity, whereas other mutants
(H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding
activity. The 5'-phosphate recognition activity of these mutants was
examined by UV cross-linking to a 5-nucleotide gap DNA where the 5'
terminus in the gap was either phosphorylated or unphosphorylated. The results indicate that Lys35 is involved in 5'-phosphate
recognition of DNA polymerase
. Finally, the dRP lyase activity of
these mutants was evaluated using a preincised apurinic/apyrimidinic
DNA. Alanine mutants of Lys35 and Lys60 are
significantly reduced in dRP lyase activity, consistent with the lower
ssDNA binding activity. More importantly, alanine substitution for
Lys72 resulted in a greater than 90% loss of dRP lyase
activity, without affecting DNA binding. Alanine mutants of
Lys68 and Lys84 had wild-type dRP lyase
activity. The triple alanine mutant, K35A/K68A/K72A, was devoid of dRP
lyase activity, suggesting that the effects of the alanine substitution
at Lys72 and Lys35 were additive. The results
suggest that Lys72 is directly involved in formation of a
covalent imino intermediate and are consistent with Lys72
as the predominant Schiff base nucleophile in the dRP lyase
-elimination catalytic reaction.
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INTRODUCTION |
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Genomic DNA is constantly exposed to various endogenous and
external environmental agents leading to DNA base loss and/or damage.
To remove such damage and retain genome stability, the base excision
DNA repair pathway has been maintained in essentially all organisms.
Base excision repair was initially described in Escherichia
coli (1) and later in mammalian cells (2). This repair pathway is
initiated by enzymatic removal of an inappropriate base or spontaneous
hydrolysis of bases through cleavage of the N-glycosyl bond
(3, 4). The resulting apurinic/apyrimidinic (AP)1 site is cleaved by a
class II AP endonuclease (5), which incises the phosphodiester backbone
5' to the AP site resulting in a 3'-hydroxyl and 5' 2-deoxyribose
5-phosphate (dRP) containing termini. To complete repair, the dRP
moiety is removed so that a single-nucleotide gap with a 3'-hydroxyl
and 5'-phosphate is generated (6, 7). DNA polymerase (
-pol)
replaces the missing nucleotide (7-9), and DNA ligase I seals the
nicked product (9, 10). These enzymatic activities should be
coordinated for efficient base excision repair.
-pol is a multifunctional enzyme consisting of an 8-kDa
amino-terminal domain with dRP lyase activity (11, 12) and a 31-kDa
carboxyl-terminal domain with nucleotidyltransferase activity (13). The
crystal and solution structures of the amino-terminal 8-kDa domain have
been determined (14, 15). The 8-kDa domain (residues 1-87) is formed
by four
-helices, packed as two antiparallel pairs. The pairs of
-helices cross one another at 50° giving them a V-like shape (15,
16). The 8-kDa domain of
-pol also contains a motif termed
"Helix-hairpin-Helix" (HhH) (17). This motif has been found in a
number of DNA repair proteins, including several DNA glycosylases and
AP lyases (18). Residues of the HhH motif have been proposed to
contribute to recognition and excision of damaged nucleotides in DNA,
as well as AP lyase chemistry (18, 19). Alignment of the HhH motifs
from
-pol and endonuclease III suggest that
-pol
Lys68 may be critically important in lyase chemistry since
mutation of the analogous lysine residue in endonuclease III,
Lys120, resulted in a dramatic reduction in
kcat (20). Furthermore,
-pol protein residues
involved in single-stranded DNA (ssDNA) binding have been identified by
NMR using chemical shift changes (15). The Helix-3-hairpin-Helix-4
motif and residues in an adjacent
-type loop form the ssDNA
interaction surface (15, 16). The x-ray crystal structure of
-pol
bound to a template-primer substrate suggested that four lysine
residues (residues 35, 68, 72, and 84) in this region of the protein
coordinate the DNA 5'-phosphate that may exist in a gapped DNA (17).
However, in structures of
-pol bound to a one-nucleotide DNA gap
only Lys35 and Lys68 coordinate the
5'-phosphate in the short gap (21). Based on information available from
the crystal and NMR structures of the 8-kDa domain and from biochemical
studies of the protein (14-16, 22), we have now conducted
site-directed mutagenesis to alter 9 residues in the 8-kDa domain of
-pol that appear to contribute key interactions to ssDNA binding,
5'-phosphate recognition in a DNA gap, and dRP lyase activity. The
results allow us to identify two critical residues, Lys35
and Lys72, for 5'-phosphate recognition and dRP lyase
chemistry, respectively.
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EXPERIMENTAL PROCEDURES |
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Materials--
Synthetic oligodeoxyribonucleotides purified by
HPLC were obtained from Operon Technologies, Inc. Unphosphorylated
oligodeoxythymidylate, (dT)16, was from Pharmacia.
[-32P]ddATP and [
-32P]ATP (3000 Ci/mmol) were from Amersham. Terminal deoxynucleotidyltransferase and
T4 polynucleotide kinase were from Promega. Human AP endonuclease and
uracil-DNA glycosylase (UDG), with 84 amino acids deleted from the
amino terminus, were purified as described (23, 24).
Mutagenesis, Expression, and Purification of the Recombinant Wild-type 8-kDa and Mutant Proteins-- Oligonucleotide site-directed mutagenesis was performed using a procedure described previously (25). Recombinant amino-terminal 8-kDa domain and the mutant proteins were overexpressed and purified as described (22).
Circular Dichroism Spectroscopy--
For CD analysis, the
wild-type 8-kDa domain of -pol and the mutants were further purified
by gel filtration on HPLC using a Bio-Sel SEC-125 (300 × 7.8 mm)
size exclusion column (Bio-Rad). Buffer consisted of 5 mM
Tris-HCl, pH 7.2, and 500 mM NaCl. The chromatogram for the
mutants was compared with the chromatogram of the highly purified
wild-type 8-kDa domain in selecting the pooled peak fraction for each
mutant. The concentrations of the mutant proteins were determined by UV
absorption at 280 nm (
280 = 5440 M
1 cm
1). CD measurements were
performed on a Jasco J715 spectropolarimeter in a 1-cm cell at
25 °C. The CD spectra were collected from 260 to 200 nm at a
resolution of 1 nm using up to 8 scans. The per residue molar
ellipticity (deg·cm2 dmol
1) was calculated
from the concentration for the 87-residue polypeptide.
5'-End Labeling--
Unphosphorylated oligodeoxyribonucleotide
(P1) was labeled by T4 polynucleotide kinase using
[-32P]ATP as described (26, 27).
Template-Primer Annealing-- Lyophilized oligonucleotides were resuspended in 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA, and the concentrations were determined from their UV absorbance at 260 nm. Template-primers were annealed as described previously (27). The sequence of the 5-nucleotide gapped DNA used was as follows.
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UV Cross-linking to Gapped DNA-- Purified wild-type 8-kDa domain or the mutant protein (1.4 µM) was mixed with the gapped DNA template-primer (0.7 µM) in a reaction mixture containing 20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA, and 5 mM MgCl2 and incubated at room temperature for 15 min. The samples were irradiated, and the photochemical cross-linked 8-kDa protein-DNA complexes were separated and analyzed as described (28). The relative activities were determined by the ability of the mutant 8-kDa domain to discriminate between a 5'-phosphorylated and unphosphorylated terminus in a gap as compared with wild-type protein. The wild-type 8-kDa binds to a 5'-phosphorylated 5-nucleotide gap 2.5-fold more readily than when the gap has a 5'-hydroxyl (i.e. amount of cross-linked complex in a 5'-phosphorylated gap/amount of cross-linked complex in a unphosphorylated gap). The loss of 5'-phosphate recognition results in equal cross-linked mutant complexes in the presence or absence of a 5'-phosphate in the gap. To quantify the cross-linked complexes, the autoradiogram was scanned on an Imager Master VDS, and the data were analyzed using ImageMaster software.
UV Cross-linking of Oligo(dT)16-- Typically, wild-type 8-kDa domain or a mutant protein (50 µM) was mixed with [32P](dT)16 (14 µM) in a 15-µl reaction mixture containing 20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA, and 5 mM MgCl2. The samples were irradiated, and the photochemical cross-linked 8-kDa protein-[32P](dT)16 complexes were separated and analyzed as described (22). To quantify cross-linking, the dried gels were scanned on a PhosphorImager 450 (Molecular Dynamics), and the data were analyzed using ImageQuant software.
3'-End Labeling--
A 49-mer oligodeoxyribonucleotide
containing uracil at position 21 was labeled at the 3'-end by terminal
deoxynucleotidyltransferase using [-32P]ddATP as
described (12).
Preparation of the dRP Lyase Substrate-- 32P-Labeled uracil containing duplex DNA (62.5 nM) was pretreated with 10 nM UDG in 100 µl of buffer containing 70 mM Hepes, pH 7.4, 0.5 mM EDTA, and 0.2 mM dithiothreitol. The reaction mixture was incubated at 37 °C. After a 20-min incubation, the reaction mixture was supplemented with 5 mM MgCl2 and 10 nM AP endonuclease, and the incubation was continued for another 20 min.
dRP Lyase Activity Assay-- dRP lyase activity was performed in a reaction mixture (10 µl) containing 50 mM Hepes, pH 7.4, 5 mM MgCl2, 2 mM dithiothreitol, and 20 nM preincised 32P-labeled AP site containing DNA. The reaction was initiated by adding 10 nM wild-type 8-kDa domain or a mutant derivative and incubated at 37 °C for 10 min. The reaction was terminated by transfer to 0-1 °C, and the DNA product was stabilized by addition of 2 M NaBH4 to a final concentration of 340 mM and incubation for 30 min on ice. The stabilized DNA products were recovered by ethanol precipitation in the presence of 0.1 µg/ml tRNA and resuspended in 10 µl of gel-loading buffer (95% formamide, 20 mM EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol). After incubation at 75 °C for 2 min, the reaction products were separated by electrophoresis in a 20% polyacrylamide gel containing 8 M urea in 89 mM Tris-HCl, 89 mM boric acid, and 2 mM EDTA, pH 8.8, and visualized by autoradiography. To quantify the product, gels were scanned on a PhosphorImager 450 (Molecular Dynamics), and the data were analyzed using ImageQuant software.
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RESULTS AND DISCUSSION |
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Structure-guided Site-directed Mutagenesis and Purification of
Mutant Proteins--
To probe the functional importance of residues in
the amino-terminal 8-kDa domain, 9 residues were selected from x-ray
(14, 17, 21) and NMR (15, 16) structural analyses and altered by
site-directed mutagenesis as described previously (25). These residues
were Phe25, His34, Lys35,
Lys60, Lys68, Glu71,
Lys72, Glu75, and Lys84. The
residues and alterations selected for mutagenesis were based on the
proposed role of each residue in DNA binding (15, 22) or putative role
in dRP lyase chemistry, as discussed in detail previously (11, 12, 28).
Briefly, the primary structure of a portion of the HhH motif (residues
55-79) in the 8-kDa domain is similar to that of the HhH motif in
E. coli endonuclease III glycosylase/AP lyase (17-20). In
addition, significant intermolecular nuclear Overhauser effects and
chemical shift changes were observed in the
1H-15N heteronuclear single quantum correlation
NMR spectra for surface-exposed residues upon addition of ssDNA (15),
including residues Phe25, Lys60,
Glu71, Lys72, and Glu75.
Lys68 is adjacent to residues showing chemical shift
changes. Lys84 is in the NMR unstructured linker region
between the 8-kDa and 31-kDa domains but is coordinated with the
5'-phosphate in one of the crystal structures, along with
Lys35, Lys60, and Lys72 (16, 17).
In the NMR structure, the adjacent flexible -loop residue
Lys35 contributes to the basal surface charge potential and
is adjacent to Lys72 and Lys68 (15). Also in
the
-loop, His34 has been shown to cross-link with ssDNA
(22) and to stack with a template strand base in the crystal structure
of
-pol bound to DNA (21).
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CD Spectra of Mutant Proteins-- The CD spectra of single mutants K35A, K68A, K72A, K84A, E71Q, and E75A, the double mutant (K68A/K72A), and the triple mutant (K35A/K68A/K72A) of the 8-kDa domain were similar to the CD spectrum of the wild-type 8-kDa domain. The comparable maximal negative ellipticities at 208 and 220 nm indicated that the overall helical structure in the mutants was similar to that seen in wild-type 8-kDa domain (Fig. 2). The differences in the absolute molar ellipticity at 220 nm for the mutants relative to wild type are due to error inherent in the protein concentration determinations. Positive ellipticity in the K35A and K72A single mutants at 240-260 nm was due to the presence of a minor DNA contamination eluting from the ssDNA cellulose column during purification. This DNA contaminant, however, is not seen in the CD spectrum of the highly purified wild-type 8-kDa protein. There is an increase in the ratio of negative ellipticity at 222 nm to negative ellipticity at 208 nm in the K68A/K72A mutant in comparison with the relative ellipticities at these wavelengths observed with wild type (i.e. more negative ellipticity at 222 nm and less negative ellipticity at 208 nm in the double mutant). The increase in the ratio of the maximal negative ellipticity at 222 nm versus 208 nm in the double mutant suggests an increase in helical structure in comparison with the wild-type 8-kDa domain. Similar results for the K35A/K68A/K72A triple mutant were also observed. On the basis of these results, we conclude that the substitutions chosen at these surface-exposed residues do not adversely affect the overall structure of the 8-kDa domain and that the effects of the mutations on ssDNA binding, 5'-phosphate recognition in gapped DNA, and dRP lyase activity are likely the result of the loss of specific side chain functionality.
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Single-stranded DNA Binding Activity--
The ssDNA binding
activity of the 39-kDa enzyme has been demonstrated to reside in the
8-kDa domain (22). The ssDNA binding activities of the wild-type 8-kDa
domain and mutant proteins were examined by an assay involving
photochemical cross-linking to oligo(dT)16 as described
previously (22). To assay ssDNA binding activity, purified protein was
mixed with [32P](dT)16 and irradiated with UV
light to covalently cross-link the bound ligand. The cross-linked
products were separated by SDS-PAGE and scored by autoradiography and
PhosphorImager scanning (Fig. 3 and Table
I). Under the conditions of the assay,
the level of cross-linking is proportional to the equilibrium
association constant, Ka (22). Quantitative analysis
of the UV cross-linked products indicated that the single mutants K35A, K60A, K68A, and F25W, and the triple mutant K35A/K68A/K72A were reduced
in ssDNA binding by approximately 60-75% compared with wild type
(Fig. 3 and Table I). The other mutants had similar ssDNA binding as
wild type. Alanine substitution for Lys35,
Lys68, and Lys60 had the weakest binding.
Interestingly, alanine substitution for Lys72 did not
affect ssDNA binding activity. In the intact enzyme, Lys72
has been shown to be a target for pyridoxal 5'-phosphate modification and was protected by dNTP binding (29). Lys72 has also been
implicated in forming a Schiff base intermediate with abasic site DNA
(12). The mutant bearing a glycine substitution for His34
was slightly reduced (25%) in ssDNA binding activity. This
histidine residue was found to be covalently UV cross-linked to ssDNA
(22), and in the crystal structure (Fig. 1), the imidazole ring was observed to stabilize the template base near the polymerase active site
by stacking interactions (21). In contrast, alanine substitution for
Lys68 reduced ssDNA binding activity by 70%. Surprisingly,
a double alanine mutation at Lys68 and Lys72
exhibited wild-type ssDNA binding activity, suggesting a compensating effect in the double mutant protein. The triple mutant, K35A/K68A/K72A, showed further reduction in ssDNA binding activity over that observed with the K68A/K72A double mutant (Fig. 3 and Table I). These results
confirm the importance of Lys35 in ssDNA binding
activity.
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5'-Phosphate Recognition in Gapped DNA--
Earlier, we had shown
that cross-linking of -pol to gapped DNA is dependent on a
5'-phosphate moiety in the gap. This DNA gap binding of
-pol was
directed by the amino-terminal 8-kDa domain (27). Additionally, the
crystal structure of
-pol in complex with gapped DNA substrates
suggests that the 5'-phosphate in the gap is coordinated either by four
lysines of the 8-kDa domain (residues 35, 68, 72, and 84) (17), or
Lys35 and Lys68 (21), as illustrated in Fig. 1.
These results were consistent with NMR studies showing protein-DNA
interactions at or near these same lysine residues, among other
residues (15). To identify the residue(s) involved in 5'-phosphate
recognition in gapped DNA, a synthetic gapped DNA substrate was formed
by annealing two 17-residue oligonucleotides (designated P1
and P2) to a 39-residue template (T) creating a
5-nucleotide gap between the 3'-hydroxyl of P1 and the
5'-phosphate or 5'-hydroxyl of P2 (see Fig.
4B and "Experimental
Procedures"). This DNA substrate was incubated with wild-type or
mutant proteins of the 8-kDa domain, and the complex was then
photochemically cross-linked with UV light (27). To score the
covalently cross-linked complexes, the 5'-end of the P1
oligonucleotide was 32P-labeled. After cross-linking, the
mixture was separated by SDS-PAGE, and the gel was analyzed by
autoradiography (Fig. 4A and Table I). The results show that
cross-linking between the 8-kDa domain, template (T), and primer
(P1) is strongly influenced by the phosphate group on the
5'-end of P2 as previously demonstrated (27). Whereas the
Lys35 alanine mutant showed a strong decline in
5'-phosphate recognition activity, alanine substitutions of
Lys60, Lys68, or Lys72 retained the
recognition activity (Fig. 4A and Table I). The E75A mutant
also displayed a diminished ability to discriminate between the
phosphorylated and unphosphorylated gaps. The lower amount of
cross-linking for the K60A and K68A mutants is consistent with the
lower ssDNA binding activity of these mutants, but both mutants
retained the 5'-phosphate recognition activity of wild type (Fig.
4A and Table I). Both NMR and crystallography data suggested
that Lys35 and Lys68 are sites for 5'-phosphate
contact (15, 17, 21). Our results on 5'-phosphate recognition with the
K35A mutant support the conclusion that Lys35 coordinates
the 5'-phosphate group, whereas Lys68 does not.
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dRP Lyase Activity--
To examine the dRP lyase activity of
wild-type and mutant enzymes, we utilized a 49-residue oligonucleotide
duplex DNA, which contained a uracil residue at position 21. The
uracil-containing strand was 3'-end labeled with
[-32P]ddAMP and annealed to its complementary DNA
strand. To prepare DNA substrate for the dRP lyase reaction, the
32P-labeled duplex DNA was pretreated with UDG and AP
endonuclease. Thus, the resulting DNA substrate contains a 5'-dRP group
and a 32P-labeled ddAMP residue at the 3'-end of the
downstream DNA strand (Fig.
5B). Wild-type and mutant
enzymes were incubated with this pretreated 32P-labeled
duplex DNA, and at the end of each reaction period the DNA product was
stabilized by NaBH4. The release of 5'-dRP from the
32P-labeled substrate was determined by the appearance of a
new radioactive electrophoretic band migrating approximately one-half nucleotide faster than the substrate (Fig. 5A). Results of
dRP lyase activity of alanine substitutions are shown (Fig.
5A), but the results of all the mutants have been summarized
(Fig. 5C and Table I). Our results indicate that
Lys68 retained wild-type dRP lyase activity when mutated to
alanine. We had previously considered that this residue was a candidate nucleophile involved in Schiff base formation during the lyase reaction, based on sequence alignment with endonuclease III (28). Mutagenesis of the corresponding lysine (residue 120) in the HhH motif
of endonuclease III strongly reduced AP site lyase activity (20). In
contrast, our results indicate that Lys68 is not a
candidate for Schiff base formation in the dRP lyase reaction catalyzed
by the 8-kDa domain of
-pol. While mutants E75A, K35A, H34G, and
K60A retained approximately 40-75% dRP lyase activity, the K72A had
less than 10% activity of wild type (Fig. 5C). There was no
further decline in dRP lyase activity with the K68A/K72A double mutant
over that of the K72A single mutant, suggesting that a Schiff base
nucleophile role for Lys68 in the K72A mutant is
unlikely.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Pathology, University of South Alabama, Mobile, AL 36617.
To whom correspondence should be addressed:
Laboratory of Structural Biology, National Institute of Environmental
Health Sciences, 111 T.W. Alexander Drive, Bldg. 101, Rm. B246,
Research Triangle Park, NC 27709. Tel.: 919-541-3267; Fax:
919-541-2260; E-mail: wilson5{at}niehs.nih.gov.
1
The abbreviations used are: AP,
apurinic/apyrimidinic; -pol, DNA polymerase
; dRP, 2'-deoxyribose
5'-phosphate; HhH, Helix-hairpin-Helix; HPLC, high pressure liquid
chromatography; UDG, uracil-DNA glycosylase; ssDNA, single-stranded
DNA; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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