From the Lineberger Comprehensive Cancer Center and the Department of Biochemistry & Biophysics, University of North Carolina Medical School, Chapel Hill, North Carolina 27599-7295
Received for publication, September 9, 2002, and in revised form, December 31, 2002
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ABSTRACT |
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NaeI endonuclease contains a
10-amino acid region with sequence similarity to the active site
KXDG motif of DNA ligase except for leucine (Leu-43)
in NaeI
(43LXDG46). Changing Leu-43 to
lysine abolishes the NaeI endonuclease activity and
replaces it with topoisomerase and recombinase activities. Here we
report the results of substituting Leu-43 with alanine, arginine,
asparagine, glutamate, and histidine. Quantitating specific activities
and DNA binding values for the mutant proteins determined the range of
amino acids at position 43 that alter NaeI mechanism. Substituting alanine, asparagine, glutamate, and histidine for Leu-43
maintained endonuclease activity, but at a lower level. On the other
hand, substituting positively charged arginine, like lysine at position
43, converted NaeI to a topoisomerase with no observable
double-strand cleavage activity. The specific activities of
NaeI-43K and NaeI-43R and their relative
sensitivities to salt, the topoisomerase-inhibiting drug
N-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide (amsacrine) and single-stranded DNA showed that the two activities are
similar. The effect of placing a positive charge at position 43 on
NaeI structure was determined by measuring (for
NaeI and NaeI-43K) relative susceptibilities to
proteolysis, UV, circular dichroism spectra, and temperature melting
transitions. The results provide evidence that a positive charge at
position 43 induces dramatic changes in NaeI structure that
affect both the Endo and Topo domains of NaeI. The
identification of four putative DNA ligase motifs in NaeI
leads us to speculate that structural changes that superimpose these
motifs on the ligase structure may account for the changes in activity.
NaeI endonuclease
(NaeI)1 is a
prototype for the type IIe (enhancer) (1, 2) restriction endonucleases,
so named (3) because they require interactions with an enhancing DNA
sequence. One DNA sequence acts as enhancer to induce cleavage of the
other sequence (4-7). In solution, NaeI protein is a 70-kDa
homodimer (7) composed of two 317-amino acid polypeptides (8, 9) that
recognize and cleave at the arrow, the DNA sequence 5'-GCC NaeI position 43 is located near the C terminus of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
GGC-3' using only Mg2+ as a cofactor. Two-site binding gives
NaeI a specificity of DNA recognition
~104-fold better than single-site-binding proteins that
recognize similar sized sequences (3). The two-site binding loops out intervening DNA sequences (5), hinting at more complicated functions
than monofunctional DNA cleavage. Substituting lysine for leucine at
position 43 (L43K) in NaeI endonuclease abolishes restriction endonuclease activity and in its place gives topoisomerase and recombinase activities (10). In addition, substitution L43K results
in a preference for binding of single-stranded DNA and a sensitivity to
salt and intercalative topoisomerase-inhibiting drugs, such as
N-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide (amsacrine), which is lacking in restriction endonucleases but characteristic of topoisomerases (11, 12).
-helix
H2, which is part of the central hydrophobic core of the Endo domain (Ref. 13 and Fig. 1). Unlike the
structures of most restriction endonucleases, NaeI contains
two separate domains, both of which bind DNA (14). The N-terminal, Endo
domain contains the restriction endonuclease cleavage motif found in
restriction enzymes as well as repair nucleases mutH, Vsr, and
exonuclease (14-17) and transposases (18). The C-terminal, Topo domain
contains a CAP motif also found in topoisomerases IA and II (19).
Position 43 is positioned at the bottom edge of the Endo domain almost
between the Endo and Topo domains and at the boundary separating the
two NaeI monomers (Fig. 1) The two Leu-43 residues lie
toward the center of the NaeI dimer ~15 Å apart.
View larger version (80K):
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Fig. 1.
NaeI-DNA crystal structure
(14). Wild-type (-L43) NaeI protein dimer is
shown as red and gold ribbons. DNA is
shown in green. Leucine at position 43 is shown as
cyan ball and stick and oversized
for clarity. Endo (N-terminal) and Topo (C-terminal) domains are
indicated.
The NaeI position 43 lies within a 10 amino acid region with
similarity to the conserved, active-site KXDG motif for DNA
ligases, RNA ligases, and RNA-capping enzymes, which together make up
the nucleotidyl transferase superfamily (for discussions, see Ref. 20).
Nucleotidyl transferase catalysis involves three steps. First, the
ligase is activated by the formation of a covalent protein-AMP
intermediate with the AMP linked to the -amino group of lysine by a
phosphoramidate bond. The conserved lysine in the sequence
KXDG forms the adenylated intermediate using the high energy
cofactors ATP (generally found in eukaryotes, viruses, and
Archaebacteria) and NAD+ (generally found in Eubacteria).
Second, the AMP moiety is transferred from lysine to the 5'-phosphate
at the nicked DNA. Finally, the DNA-free ends are joined in an
enzyme-dependent reaction with loss of AMP. NaeI
has leucine instead of the essential lysine at position 43 (43LXDG46). The topoisomerase
activity of NaeI-43K is possibly the result of activating a
cryptic ligase active site and thereby coupling restriction
endonuclease cleavage with ligation. NaeI forms a covalent
intermediate with a cleaved substrate (10), which may serve as the high
energy intermediate needed for ligation, as is the case with the
topoisomerases and recombinases. The amino acid that covalently links
NaeI to its DNA substrate has not been identified. There is
no similarity between the folds of NaeI (13, 14) and DNA
ligase (21-24). Moreover, the KXDG-like motif in NaeI lies away from the endonuclease metal-binding site
necessary for cleavage. Thus, the transformation to topoisomerase
activity in L43K implies a conformational change in the ES complex that results in the KXDG-like motif lying closer to the
phosphodiester scissile bond; the endonuclease fold may be altered to
mimic aspects of the ligase fold found at the active sites of the DNA ligases.
To learn whether lysine at position 43 is unique in its ability to give
NaeI topoisomerase activity, we substituted alanine, asparagine, glutamate, histidine, and arginine. We also substituted lysine for leucine at position 40. Alanine has a small, nonpolar, amino
acid side chain. Asparagine has an uncharged, amide-bearing polar side
chain. Like lysine, histidine, arginine, and glutamate all have
charged, polar side chains depending on the pH. We report that alanine,
asparagine, glutamate, and histidine at position 43 maintained
NaeI wild-type endonuclease activity when substituted at
position 43, but with significant decreases in DNA cleavage. Substitution with arginine, however, resulted in topoisomerase activity
identical to that of NaeI-43K. NaeI and
NaeI-43K susceptibility to protease, UV-circular dichroism
(CD) spectra, and CD temperature melting transitions were determined.
The results provide the first evidence that placing a positive charge
at position 43 causes a dramatic change in NaeI folding. The
finding of additional putative ligase motifs within the NaeI
sequence offers a possible rationale for how the conformational changes
may alter NaeI activity.
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EXPERIMENTAL PROCEDURES |
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Materials--
Escherichia coli strain CAA1
(Fe14
(mcrA
) lacY1 or D
(lac)6 SupE44 galK2 galT22 mcrA rfbD1
mcrBa hsd (rk
mk+)
M·MspI+) and plasmid pNEB-786, containing the
NaeIR gene, were obtained from New England Biolabs. Plasmid
pMAL-C2 and amylose resin were purchased from New England Biolabs.
Substrate pBR322 was purchased from Promega Corp. Amsacrine, a
DNA-intercalating drug that inhibits topoisomerase activity was
purchased from Topogen Inc. Oligodeoxyribonucleotides (oligonucleotides) were synthesized by the Nucleic Acid Core Facility at UNC. Cellulose phosphate, sp-Sepharose, and heparin resins were purchased from Sigma. Cognate
(dTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT) and noncognate
(dTTTCTCGCCACGTTCGAAGAATTTCCCCGTCAAGCT)
oligonucleotides were annealed to their complements to yield DNA
fragments, which were gel purified.
NaeI Substitutions-- The NaeIR gene was subcloned into the expression vector pMAL-C2, and site-directed mutagenesis was performed using the method of Clackson et al. (25). Mutated NaeIR genes were sequenced to confirm the mutation and ensure that no secondary mutations were generated.
Purification of NaeI Mutants--
To express the fusion protein,
pMAL-NaeI mutants (MBP·NaeI) were
induced with 1 mM
isopropyl--D-thiogalactoside (IPTG). Cells were
harvested by centrifugation and cell pellets resuspended in 4 volumes
of Tris column buffer (10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 5% glycerol, 1.0 mM
2-mercaptoethanol, 50 mM NaCl) with 1 mM
phenylmethylsulfonyl fluoride to inhibit serine proteases. The
resuspended cell pellet was sonicated on ice for 1 min per 10 ml
followed by centrifugation at 30,000 × g to remove
cellular debris. The supernatant was subjected to amylose resin
chromatography. The column was washed with column buffer containing 400 mM NaCl then equilibrated with column buffer containing 50 mM NaCl. Fusion protein was eluted in Tris column buffer
containing 10 mM maltose. Maltose had no effect on
NaeI and NaeI-43K activities.
NaeI-- 43R was cloned into pNEB786 (to give pNEB786-NaeI-43R) and isolated from cells containing this plasmid. Cell extracts showed topoisomerase activity absent from extracts from cells containing pNEB786. Phosphocellulose, sp-Sepharose, and heparin columns were used for purification. Protein elution was achieved by NaCl gradients in column buffer. Fractions were assayed for topoisomerase activity. Peak fractions containing Topo activity were pooled and dialyzed with column buffer containing 50 mM NaCl after each column. Heparin fractions were dialyzed against 50 mM NaCl in column buffer for storage. NaeI-43R identity was confirmed by Western analysis using an affinity-purified antibody prepared against wild-type NaeI. The final purities of the proteins was estimated to be >90% based on optical density measurements of the protein resolved by SDS-PAGE by standard procedures.
Restriction Endonuclease Activity--
Restriction endonuclease
activities relative to wild-type NaeI were determined from
measurements of steady-state DNA cleavage rates. Protein concentrations
were titrated while keeping reaction time (30 min) and DNA
concentration (500 nM) constant. Reactions were prepared in
15-µl total volume to contain 10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and bovine serum albumin (0.1 mg/ml).
Reactions were incubated at 37 °C for 30 min. Substrate was cognate
DNA radiolabeled with [-32P]ATP using T4
polynucleotide kinase. Reactions were stopped by addition of EDTA (40 nM) and glycerol (10%). Reaction products were separated
on 8% polyacrylamide gels and analyzed using a Molecular Dynamics
Storm 540 PhosphorImager. The cleaved and uncleaved gel bands were
quantified using Imagequant 5.0 from Molecular Dynamics.
Gel Mobility Shift Assays-- Single-stranded oligonucleotides were radiolabeled and annealed to complementary oligonucleotides to give probe. Protein and probe were incubated in a 20-µl volume containing 10 mM Tris-HCl (pH 8.0), 10 mM CaCl2, 20 mM NaCl, 10% glycerol, and bovine serum albumin (0.1 mg/ml). Reactions were incubated at 25 °C for 20 min. Reaction products were analyzed by PAGE (6%). Apparent KD values (defined by the protein concentrations necessary to shift half the amount of probe) were determined with cognate probe. Nonspecific binding was tested using noncognate probe.
Assay of NaeI-43R Topoisomerase Activity--
Plasmid pBR322
(11.6 nM) and NaeI-43R (0.21 µM)
were incubated at 37 °C for 30 min in 10 µl containing 10 mM Tris-HCl (pH 8.0), 20 mM NaCl (except for
the assay of NaCl dependence), 5 mM MgCl2,
bovine serum albumin (0.1 mg/ml), and 5 mM
-mercaptoethanol. Its weak binding to DNA (12) necessitated the
relatively high concentration of NaeI-43R. The DNA binding
affinity was similar to that of NaeI-43K. Reactions were
stopped by addition of SDS to 1%. Products were resolved on 1.0%
agarose gels containing 0.5 µg/ml ethidium bromide in the gel and in
the running buffer. Assays for the effects on NaeI-43R
activity of single-stranded DNA, NaCl, and amsacrine varied the
concentrations of these, as indicated.
Limited Proteolysis-- Trypsin (2.3 ng) was added to 7 µg of NaeI or NaeI-43K, and 1-µg aliquots were removed at the times indicated. Phenylmethylsulfonyl fluoride (1 mM) and 1.0% SDS were added to the aliquots, which were then heated to 100 °C for 10 min. Reaction products were resolved by SDS-PAGE (5% stacking and 15% resolving). The gel was stained with Coomassie Blue and photographed.
Size-exclusion Chromatography-- NaeI and NaeI-43K were sized relative to known molecular weight proteins using chromatography through Sephacryl S-200 resin (32-cm column) pre-equilibrated with phosphate column buffer (20 mM potassium phosphate (pH 7.4), 0.1 mM EDTA, 5% glycerol, 1.0 mM 2-mercaptoethanol, 50 mM NaCl). Protein (50 µl at 0.1 mg/ml) was loaded and eluted at 0.15 ml/min. Absorbance was monitored at 280 nm and the elution volume determined. The void volume was determined using blue dextran. Chromatography was performed with a Bio-Rad Biologic Chromatography System.
Circular Dichroism--
UV-CD spectra were measured using an
Applied Photophysics PiStar-180 spectrometer. NaeI and
NaeI-43K were extensively dialyzed into 10 mM
phosphate, pH 7.0 (buffer conditions that showed similar specific
activities to that measured using the above assay conditions). CD
spectra were measured for NaeI, NaeI-43K, and
buffer between wavelengths 185 nm and 260 nm at 25 °C. Buffer
spectrum was subtracted from NaeI and NaeI-43K
spectra. The concentrations of both NaeI and
NaeI-43K were 0.1 mg/ml as determined from absorbance
measurements at 280 nm. CD measurements were also made at a wavelength
of 208 nm while increasing the temperature from 10 to 90 °C.
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RESULTS |
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Activity of NaeI Mutants--
All of the mutants were expressed as
fusions with maltose-binding protein. The effects of substitutions at
position 43 were determined. DNA specific activities for each mutant
were determined from the slope of the line defined by amount cleaved
per amount of protein (Fig. 2). The plots
of DNA cleavage versus amount of protein were reproducible
and linear over the entire range of protein concentration (Fig. 2 and
Table I). Substitutions with alanine,
asparagine, glutamate, and histidine retained endonuclease activity.
The cleavage patterns of the fusion protein mutants were identical to
that of wild-type NaeI fused to maltose-binding protein
(MBP·NaeI). The specific activities of the mutants,
however, were reduced compared with that of MBP·NaeI. When
NaeI-43K and -43R were expressed as protein fusions with
maltose-binding protein neither restriction endonuclease activity nor
topoisomerase activity was detected. NaeI, expressed from
this vector, showed no topoisomerase activity. Therefore,
NaeI-43K and -43R were expressed in E. coli from
pNEB786 where they both demonstrated topoisomerase activities. Therefore, all of the mutants were studied as fusion proteins (MBP·NaeI-43A, 43H,
43E,
43N), except for
NaeI-43K and NaeI-43R.
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Relative DNA Binding--
The apparent KD
values were determined for NaeI mutant interactions with DNA
from the amount of protein necessary to shift half of the cognate DNA
probe during PAGE (Fig. 3 and Table I).
DNA-binding results are the average of two determinations. The apparent
KD values varied significantly from wild type only
for MBP·NaeI-43N, -43E, and -40K, which gave values of
125 ± 2, 225 ± 5, and 1250 ± 75 (nM),
respectively. The gel-shift results show a small amount of density in
the wells probably due to a small amount of aggregated protein that
binds DNA. The small amount of protein in the wells is counted in the
determination of relative KD values.
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Comparison of NaeI-43R and NaeI-43K
Activities--
NaeI-43K has topoisomerase activity rather
than endonuclease activity, which is sensitive to salt, amsacrine, and
ssDNA (10-12). Incubating similar concentrations of
NaeI-43R and NaeI-43K with pBR322 resulted in
almost identical banding ladders characteristic of topoisomerase
activity (Fig. 4A). The
differences between the ladders in Fig. 4A can be attributed
to small differences in specific activities between the two protein
preparations.
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Nicking versus relaxation by NaeI-43R was assayed to determine the relative amounts of nicked versus covalently closed, fully relaxed products produced (Fig. 4B). About half the final products were the latter. NaeI-43K results in a similar amount of nicked versus relaxed DNA (26).
The effects of varying salt concentration on the relaxation reaction
were determined (Fig. 5). The optimum
NaCl concentration was below 30 mM. At 210 mM
salt, relaxation was completely inhibited. The NaeI-43R
mutation also made NaeI sensitive to the intercalative drug
amsacrine (Fig. 5B). Inhibition of NaeI-43R Topo
activity by amsacrine was apparent at a concentration of 5 µM with near total inhibition at 10 µM.
Single-stranded DNAs, containing the NaeI cognate or
noncognate sites, were tested for their ability to inhibit
NaeI-43R activity. Nearly complete inhibition of
NaeI-43R occurred with 10 nM ssDNA with or
without cognate recognition sequence (Fig. 5, C and
D). The results are identical to those for
NaeI-43K.
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Effect of L43K Substitution on NaeI Structure--
To determine
the effect of a positive charge at position 43 on NaeI
structure, NaeI and NaeI-43K conformations were
probed using size-exclusion chromatography, limited proteolysis, and circular dichroism. NaeI-43K elutes by size-exclusion
chromatography at the same volume as NaeI, indicating that
it is a dimer of about 70 kDa (Fig. 6).
Ultracentrifugation (27), gel filtration (7), and crystallization (13,
14) show that the preferred structure of NaeI in solution is
a dimer. Trypsin was used to probe the domain structure of
NaeI-43K relative to NaeI. The sites accessible to trypsin cleavage were clearly different between NaeI-43K
and NaeI (Fig. 7).
NaeI showed stable domains at molecular sizes of 16 and 19 kDa. NaeI-43K showed no stable domains and a significantly different initial banding pattern from NaeI at 5-min
digestion with trypsin (Fig. 7). Additionally, NaeI was more
resistant to trypsin cleavage than NaeI-43K.
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The protease digestion experiments implied conformational differences
between NaeI and NaeI-43K. To confirm this, we
measured the UV CD spectra of both proteins. CD measurements showed
distinct differences between the CD spectra of NaeI and
NaeI-43K (Fig. 8A).
Most notably, the minima between 205 and 230 nm were larger for
NaeI-43K, with a distinct difference at the -helical
characteristic wavelength of 222 nm. The CD curves were reproducible
and overlapped above
of 250 nm, which indicated no significant
concentration differences. CD was also used to monitor the temperature
melting profiles of the two proteins to determine their relative
Tm values. The thermal transition point was
determined at
of 208 nm, which is within the wavelength area where
the CD values are most sensitive to protein structure (28).
NaeI and NaeI-43K gave well defined melting
points of 56 ± 0.3 °C and 59 ± 0.3 °C, respectively
(Fig. 8B). The melting point values are the averages of two
determinations.
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Visual Inspection of the NaeI Amino Acid Sequence--
Initial
inspection of the NaeI sequence led to the discovery of a 10 amino acid sequence in NaeI with similarity to motif I of
DNA ligase, the KXDG motif (10). Visual inspection, taking into consideration the secondary structures of the DNA ligase motifs,
identified three additional regions of NaeI protein sequence (Fig. 9) with similarity to three
additional motifs that define the DNA ligase enzyme family (20-24,
29). Fig. 9 shows the four ligase motifs for several of the DNA ligases
and for NaeI. The NaeI amino acid sequence
regions are shown at the top of Fig. 9 in bold. Amino acids
in the DNA ligase motifs in the same exchange group (30) as the
corresponding amino acid in NaeI are shown in
bold and underlined.
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DISCUSSION |
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NaeI Position 43-- Substituting lysine for leucine at position 43 abolishes NaeI endonuclease activity and replaces it with topoisomerase and recombinase activities (10). Alanine, arginine, asparagine, glutamate, and histidine were substituted at position 43 and lysine at position 40 and the activities of the respective N-terminal MBP fusion proteins quantitated to test the chemical characteristics that lead to topoisomerase activity. All substitutions, except arginine and lysine substituted at position 43, retained sequence-specific endonuclease activity, albeit at lower levels (Table I). Substitution of alanine at position 43 retained the most cleavage activity, whereas inserting lysine at position 40 and the negatively charged glutamate at position 43 reduced cleavage ~33- and 100-fold, respectively. Alanine and histidine substitutions at position 43 retained wild-type levels of DNA binding. Substituting lysine for leucine at position 40 and glutamate for leucine at position 43, however, reduced DNA binding 125-fold and 23-fold, respectively. Deletion of the entire Endo domain only reduces DNA binding for NaeI 8-fold (27) because of the strong DNA binding of the Topo domain (6, 14, 27). Thus, placing a charge within the core hydrophobic region of the Endo domain appears to have effects that reach beyond the local environment of the Endo domain.
NaeI-43K lacks activity when expressed either as an N-terminal or as a C-terminal MBP fusion protein (not shown). NaeI-43R fused at its N terminus to MBP also lacks activity. The effect of fusion at the C terminus was not determined. When expressed without MBP, on the other hand, both mutant proteins gave identical topoisomerase activities. NaeI has endonuclease activity either when expressed alone or as an N-terminal MBP fusion protein (27). No activity is recovered from NaeI expressed as a C-terminal fusion protein. The apparent loss of activity when fused at the C terminus can be rationalized from the crystal structure of the NaeI-DNA complex (14): The C terminus of each NaeI monomer lies over the DNA binding pocket. A bulky protein fused in this position would block access of DNA to the NaeI Topo domain. On the other hand, the NaeI N terminus lies on the outside of NaeI away from the binding faces of the protein (14). Thus, the loss of activity when MBP is fused to the N terminus of NaeI-43K and -43R, but not when fused to the N terminus of NaeI, suggests an altered structure for NaeI-43K and -43R.
NaeI-43R Has Topoisomerase Activity--
NaeI-43R and
NaeI-43K were found to be similar with respect to specific
activity, NaCl dependence, amsacrine inhibition, and ssDNA inhibition.
Amsacrine and ssDNA have no affect on NaeI, but inhibited
the topoisomerase activities of NaeI-43K and
NaeI-43R, demonstrating the equivalency of the L43K and L43R
substitutions. To probe whether introducing a positive charge into the
NaeI hydrophobic core could explain the switch to
topoisomerase activity, lysine was substituted for leucine at position
40 in NaeI. Leucine 40 lies in the same -helix, H2, as
leucine 43 and is also part of the hydrophobic core of NaeI.
Substitution of lysine for Leu-40 of NaeI decreased
NaeI endonuclease activity, relative to wild type, but did
not produce topoisomerase activity (Table I). The decrease in binding
caused by L40K was similar to that seen when a negative charge (on
glutamate) was inserted at position 43. The result implies that the
topoisomerase activities in NaeI-43K and NaeI-43R
depend on chemistry that involves more than the disruption caused by
the introduction of a positive charge into the hydrophobic core. It is
also possible, however, that a positive charge placed at position 40 causes a different disruption to folding than when placed at position 43.
Positive Charge at Position 43-- The addition of a positive charge in the form of lysine or arginine is sufficient to convert NaeI from a restriction endonuclease to a topoisomerase. NaeI position 43 is located near the interface between the two subunits that compose the homodimer (Fig. 1). The addition of a positive charge could interfere with NaeI dimerization, but size-exclusion chromatography demonstrated that NaeI-43K is a dimer. Limited proteolysis of NaeI shows two distinct and stable domains as products (Ref. 27 and Fig. 7). Limited proteolysis of NaeI-43K showed a very different initial pattern of digestion products (Fig. 7), indicating that in NaeI-43K versus NaeI, different arginines and lysines are accessible to trypsin. It is clear from the proteolysis results that NaeI-43K is less stable to trypsin than NaeI. At 90 min, a large portion of full-length NaeI remained undigested, whereas the full-length NaeI-43K was almost completely digested in 60 min and by 90 min showed no domain remaining resistant to trypsin. The most resistant band from digestion of NaeI-43K with trypsin was at 17 kDa. Peptide sequencing showed that this product and the 17-kDa product of NaeI (27) come from the Endo domain. Thus, the L43K substitution, which lies in the Endo domain, has significant effect on trypsin digestion of both the Topo and Endo domains. This implies significant refolding of the protein.
The protein UV-CD spectrum is determined by the protein conformation
(28). The UV-CD spectra of NaeI and NaeI-43K
indicate significant structural differences (Fig. 8A). The
CD spectrum for NaeI-43K has the classical minima at 208 and
222 nm indicative of significant -helical structure (31), whereas in
NaeI they are not as prominent. The CD spectra were analyzed
using the computer program Continll (28). NaeI-43K showed
higher
-helical content than wild-type NaeI: 40 versus 15%
-helix, 33 versus 42%
-sheet, 17 versus 24% turn, and 10 versus 20%
unordered, respectively. The errors associated with these values are
all better than ± 1% as determined from the best-fit results. CD
analysis of protein structure is not very accurate. Here, the CD
results underestimate the amount of NaeI
-helix; the
crystal structure of NaeI shows that ~30% of the protein
is
-helical. We assume that the
-helical content of
NaeI-43K was underestimated to the same extent. Thus, by CD
analysis, NaeI-43K has an altered structure relative to wild-type NaeI.
The presence of the positive charges in NaeI-43K and
NaeI-43R could destabilize the proteins. To measure relative
stability, CD measurements at of 208 nm as a function of increasing
temperature were used to monitor NaeI and
NaeI-43K temperature denaturation (Tm) profiles (Fig. 8B).
NaeI-43K was more heat-stable. The 3 °C increase in
Tm value for NaeI-43K relative to
NaeI was surprising considering the location of position 43 in the hydrophobic core. L43K substitution causes a significant change
in NaeI structure. The change may move lysine 43 out of the
hydrophobic core so that it can participate in electrostatic
interactions that help stabilize NaeI-43K. We speculate that
the change in structure is required for the switch from endonuclease to
topoisomerase activity.
Changes in Structure and Activity-- It is reasonable to assume that a significant refolding of NaeI is necessary to convert its activity from endonuclease to that of topoisomerase and recombinase. If this is true, then the significant global conformational changes demonstrated here may be the result of this refolding. How the changes in protein folding lead to the changes in protein activity is unknown. We can speculate about a possible mechanism, however, based on sequence homologies found by visual comparisons of the NaeI protein sequence with the sequences of the DNA ligases (Fig. 9). There are five conserved nucleotidyl transferase motifs that define the ligase/capping enzyme superfamily (20-24, 29). The five collinear sequence elements, designated nucleotidyl transferase motifs I, III, IIIa, IV, and V, are conserved in ATP-dependent DNA ligases, mRNA-capping enzymes, and NAD-dependent DNA ligases. Four of five sequence motifs were detected in NaeI by visual inspection (Fig. 9). The NaeI sequences are collinear with the conserved nucleotidyl transferase motifs, and the sequence distances between the NaeI sequences are approximately within the ranges predicted from that between the corresponding nucleotidyl transferase motifs. The motifs are spread over both domains of NaeI, and the NaeI structure cannot be superimposed on the DNA ligase structure. It is interesting to speculate that the refolding of NaeI caused by the placement of a positive charge at position 43 causes at least some of these motifs to superimpose. Active site residues in NaeI bind divalent metal, which stabilizes the DNA binding and the pentavalent transition state. DNA ligase motif III contains two conserved residues: one is conserved among all the DNA ligases, the other is conserved among the ATP ligases (Fig. 9). The two conserved amino acids in motif III contribute to the third (nick-joining) step of DNA ligation. For example, in Chlorella virus DNA ligase, Asp-65 and Glu-67 enhance the rate of step 3 phosphodiester formation by 20- and 1000-fold, respectively (32). Asp-65 overlaps with NaeI Asp-86, an essential metal ion-binding amino acid in NaeI (Fig. 9), which enhances DNA cleavage by 50-fold.2 Mutations of Glu-88 in NaeI have not been studied because this position is not conserved among restriction enzymes. Study of the effects on topoisomerase activity of mutation of NaeI-43K amino acids with similarity to the ligase motifs shown in Fig. 9 are in progress. The presence of additional ligase motifs in NaeI would imply homology with the DNA ligase family and support the notion that NaeI topoisomerase activity arises through linkage of DNA cleavage and ligation activities through the NaeI-DNA covalent intermediate (10).
Comparison with Nucleotidyl Transferases-- The nucleotidyl transferase KXDG-like motif in NaeI-43K appears to provide another example of the motif retaining some activity after substitution of the active site KXDG lysine with arginine but not when substituted with other amino acids. Two nucleotidyl transferases that manifest similar behavior are vaccinia virus RNA-capping enzyme and T4 RNA ligase (33, 34). Active-site lysine to arginine substitution in vaccinia virus RNA-capping enzyme gives low overall activity, whereas other mutations give no activity (33). The equivalent substitution in T4 RNA ligase gives intermediate levels of activity in all three steps of nucleotidyl transfer (34), whereas asparagine substitution gives no activity in any of the three steps (34).
NaeI-43K and NaeI-43R can catalyze many cycles of
DNA relaxation in the absence of ATP and NAD+ (10).
Therefore, they do not require adenylation to relax DNA. Rather they
form a covalent enzyme-DNA complex (10). The energy from the
covalent complex is used for ligation, replacing the need for an
enzyme-adenylate intermediate. This suggests that NaeI-43K
and -43R require only the third step of the nucleotidyl transferase
reaction, strand closure, for topoisomerase activity. Studies of
vaccinia virus RNA-capping enzyme (33), T4 RNA ligase (34), and
Chlorella virus DNA ligase (35) show that the KXDG lysine
contributes to strand closure. In T4 RNA ligase, substitution of the
active-site lysine with asparagine inactivates the strand-closure step
(34). Lysine is not absolutely required for this step by RNA-capping
enzyme and DNA ligase but contributes 16-fold to the rate of closure
(35). Thus, the importance of the active site lysine for strand closure
may vary with the protein being considered. In NaeI,
arginine and lysine substitutions at position 43 contribute a positive
charge that alters the structure of NaeI and gives it the
ability to catalyze strand closure of newly cleaved substrate. The
result of this cleavage and ligation, when linked by the enzyme-DNA covalent intermediate, is relaxation of supercoiled DNA.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM52123.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.
To whom correspondence should be addressed: Lineberger
Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295. Tel.: 919-966-8208; Fax: 919-966-3015; E-mail: mdtopal@ med.unc.edu.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M209192200
2 K. Carrick and M. Topal, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: NaeI, NaeI endonuclease; CAP, catabolite-activating protein; ES, enzyme-substrate complex; CD, circular dichroism; oligonucleotides, oligodeoxyribonucleotides; Topo, topoisomerase; MBP, maltose-binding protein; ssDNA, single-stranded DNA; amsacrine, N-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide.
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