Sequence-selective and hydrolytic cleavage of DNA by zinc finger mutants

Article abstract of DOI:10.1021/ja045663l

We have reported the successful conversion of the structural zinc site in zinc finger peptides to a functional zinc site. A series of resulting zinc finger mutants exhibit the hydrolytic ability of the activated ester depending on the coordination geometr

Full text of DOI:10.1021/ja045663l

Published on Web 11/03/2004
Sequence-Selective and Hydrolytic Cleavage of DNA by Zinc Finger Mutants
Akiko Nomura and Yukio Sugiura*
Institute for Chemical Research, Kyoto UniVersity, Uji, Kyoto 611-0011, Japan
Received July 19, 2004; E-mail: sugiura@scl.kyoto-u.ac.jp
The molecules that can hydrolyze the DNA phosphoester at
specific positions are valuable tools in biotechnology, thus facilitat-
ing DNA manipulation in a variety of applications. Although many
naturally occurring nucleases have been utilized in the laboratory
for this purpose, designing artificial nucleases is one of the most
interesting current topics in the field of protein engineering because
artificial nucleases could expand a set of recognizable DNA
Figure 1. Zinc finger mutant peptide and protein. (a) The second zinc
finger mutant zf(HHHH) (Zn‚1). Ligands of the zinc ion, cationic amino
acids, and anionic amino acids are denoted by bold black, red, and blue
sequences beyond the limited sequences of currently available
nucleases. Most of the artificial nucleases so far reported have been
constructed by linking DNA binding moieties with DNA cleavage
moieties.1,2 For example, Ni(II)-peptide complexes1a,3 have been
attached as a DNA cutter at the terminus of DNA recognition
proteins or molecules.4 The other fascinating strategy for artificial
nucleases is to construct a hydrolytic active site in the DNA binding
peptides; for example, novel types of artificial nucleases have been
developed based on HTH and EF-hand DNA binding peptides by
introducing lanthanide ions in the original calcium binding site.5
We recently succeeded in constructing zinc finger peptides with
hydrolytic ability by mutating amino acid residues coordinated to
the zinc ion.6 Since zinc finger motifs have been known to bind to
the major groove of the DNA duplex in a sequence-specific
fashion,7 we expected that our zinc finger mutants would function
as a novel type of artificial nuclease with high sequence specificity
for DNA duplexes. In addition, DNA recognition by the zinc finger
motifs is expected to cover a wide range of DNA sequences,
compared to restriction enzymes, because zinc finger motifs bind
to asymmetric sequences while the DNA sequences recognized by
the restriction enzymes must be palindromic. In this context, we
have studied the expansion of recognizable DNA sequences by zinc
finger motifs (e.g., nine-tandem zinc finger proteins that can bind
unusually longer DNA sequences8 or R-helix-exchanged zinc finger
peptides that can bind to A/T-rich sequences instead of the original
G/C-rich sequences).9 Furthermore, the efforts to realize the DNA
recognition of a wide variety of sequences by zinc finger motifs
have been also enthusiastically pursued by other groups.10 Aiming
to create peptide-based biochemical tools exhibiting a site-specific
DNA cleavage ability, in this study, we examined the ability of
the zinc finger mutant peptides for sequence-selective DNA
hydrolysis.
First of all, we examined the ability of a zinc finger mutant
zf(HHHH) (Zn‚1),6 whose sequence is based on the second finger
in the three-tandem zinc finger protein Sp1 (Figure 1a), to hydrolyze
bis(4-nitrophenyl) phosphate (BNP) as a model compound for
DNA.11 It was revealed that Zn‚1 catalyzed the hydrolysis of BNP
to afford 4-nitrophenolate and 4-nitrophenyl phosphate.12 The
estimated second-order rate constant, k, was notably higher than
that observed for the zinc-cyclen complex, known to be an
excellent functional model for hydrolytic zinc enzymes,11a-c,13 by
2 orders of magnitude under identical conditions (Table 1). This
result encouraged us to examine the DNA hydrolytic ability of Zn‚
1. We used a supercoiled plasmid DNA, pUC19GC, as a substrate
since pUC19GC contains a GC box to which Sp1 specifically
letters, respectively. (b) The three-tandem zinc finger protein (Zn‚2).
Table 1. BNP Hydrolysis Rate Constant, k, by the Zinc
Complexesa
1
catalyst
k
×
10-5 (M-1 s-
)
Zn‚1
apo-form of Zn‚1
zinc-cyclen
502 ( 24.5
65.0 ( 5.28
7.58 ( 0.696
a The [catalyst] ) 10-20 mM, [BNP]0 ) 2-6 mM in 20 mM HEPES
buffer (pH 7.5) containing 0.1 M NaCl and 3.5% THF at 37 °C.
Figure 2. Ionic strength dependence of the plasmid DNA cleavage by Zn‚
1. The reactions were carried out at 37 °C for 96 h. [Zn‚1] ) 2 µM,
[pUC19GC] ) 57.4 nM (0.154 mM bp). Lane 1, 0 mM NaCl; lane 2, 3
mM NaCl; lane 3, 8 mM NaCl; lane 4, 18 mM NaCl; lane 5, 48 mM NaCl;
lane 6, 98 mM NaCl.
binds.14 DNA cleavage converts the supercoiled plasmid DNA (form
I) to the nicked circular form (form II) and then to the linear form
(form III). Despite the inactivated phosphoester, Zn‚1 converted
pUC19GC to form II but not to form III (Figure S1 of the
Supporting Information). The cleavage efficiency linearly increased
as the zinc concentration increased and reached a plateau at the
point of 1 equiv of zinc ion (Figure S2 of the Supporting
Information). These results emphasize that the zinc ion plays a
critical role in DNA hydrolysis.15 However, the hydrolytic reactivity
decreased along with an increase in the ionic strength (Figure 2),
which suggests that the positively charged zinc finger peptide binds
with the phosphate of DNA, rather than with the DNA bases, via
electrostatic interactions. The same hydrolytic efficiency of Zn‚1
for pUC19, which contains no GC box, as for pUC19GC indicates
nonselective binding of Zn‚1 with DNA (Figure S3 of the
Supporting Information).
Tandem alignment of zinc finger mutants, like the native Sp1,
should be a strategy to achieve selective binding at the GC box by
the zinc finger mutants because repeated zinc finger motifs show
a drastic enhancement in DNA affinity compared with that of a
single zinc finger motif.16 We already reported that a three-tandem
zinc finger protein (Zn‚2, Figure 1b), where two of the cysteine
ligands in each finger motif were replaced by histidine ligands,
retains a high affinity for the DNA duplex containing a GC box,
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J. AM. CHEM. SOC. 2004, 126, 15374-15375
10.1021/ja045663l CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
mutant Zn‚2. Interestingly, at a high ionic strength, Zn‚2 converted
the plasmid DNA to the form III. The combination of the present
strategy and the expansion of recognizable DNA sequences by zinc
finger motifs would provide a wide range of artificial nucleases.
Additionally, this hydrolytic activity may be the reason an H4-type
zinc finger protein does not exist in nature. Detailed mechanistic
studies about the site-specific hydrolysis of DNA and the construc-
tion of a family of artificial nucleases based on the zinc finger motif
are currently ongoing in our laboratory.
Figure 3. Cleavage of the plasmid DNA by Zn‚2. The reactions were
carried out with 57.4 nM (0.154 mM bp) DNA and 6 µM Zn‚2 in 5 mM
HEPES buffer (pH 7.5) at 37 °C for 48 h. Lanes 1-4, pUC19, and lanes
5-9, pUC19GC: lanes 1 and 6, 0 mM NaCl; lanes 2 and 7, 50 mM NaCl;
lanes 3 and 8, 200 mM NaCl; lanes 4 and 9, DNA only; lane 5, HindIII-
treated DNA.
Acknowledgment. This study was supported in part by Grants-
in-Aid for the COE Projects “Element Science” and “Kyoto
University Alliance for Chemistry”, and Scientific Research from
the Ministry of Education, Culture, Sports, Science, and Technol-
ogy.
Supporting Information Available: Experimental details and
results of DNA hydrolysis. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) For reviews, see: (a) Long, E. C. Acc. Chem. Res. 1999, 32, 827-836.
(b) Sreedhara, A.; Cowan, J. A. J. Biol. Inorg. Chem. 2001, 6, 337-347.
(c) Franklin, S. J. Curr. Opin. Chem. Biol. 2001, 5, 201-208.
(2) For recent examples, see: (a) Sissi, C.; Rossi, P.; Felluga, F.; Formaggio,
F.; Palumbo, M.; Tecilla, P.; Tonioli, C.; Scrimin, P. J. Am. Chem. Soc.
2001, 123, 3169-3170. (b) Bibikova, M.; Carroll, D.; Segal, D. J.;
Trautman, J. K.; Smith, J.; Kim, Y. G.; Chandrasegaran, S. Mol. Cell.
Biol. 2001, 21, 289-297. (c) Copeland, K. D.; Fitzsimons, M. P.; Houser,
R. P.; Barton, J. K. Biochemistry 2002, 41, 343-356. (d) Boseggia, E.;
Gatos, M.; Lucatello, L.; Mancin, F.; Moro, S.; Palumbo, M.; Sissi, C.;
Tecilla, P.; Tonellato, U.; Zagotto, G. J. Am. Chem. Soc. 2004, 126, 4543-
4549.
(3) (a) Huang, X.; Pieczko, M. E.; Long, E. C. Biochemistry 1999, 38, 2160-
2166. (b) Ananias, D. C.; Long, E. C. Inorg. Chem. 1997, 36, 2469-
2471.
Figure 4. Cleavage of 37 bp DNA by Zn‚2. The reactions were carried
out with 10 µM Zn‚2 in 5 mM HEPES buffer (pH 7.5) containing 75 ng/
µL poly(dI-dC) at 37 °C for 60 h. G + A and C + T are Maxam-Gilbert
sequencing lanes. The GC box sequences are highlighted by light blue (G-
strand) and yellowish green (C-strand). The numbers describe the positions
of the base pairs.
(4) (a) Mack, D. P.; Iverson, B. L.; Dervan, P. B. J. Am. Chem. Soc. 1988,
110, 7572-7574. (b) Nagaoka, M.; Hagihara, M.; Kuwahara, J.; Sugiura,
Y. J. Am. Chem. Soc. 1994, 116, 4085-4086. (c) Harford, C.; Narindra-
sorasak, S.; Sarkar, B. Biochemistry 1996, 35, 4271-4278.
(5) (a) Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem.
2001, 40, 1982-1984. (b) Kovacic, R. T.; Welch, J. T.; Franklin, S. J. J.
Am. Chem. Soc. 2003, 125, 6656-6662.
(6) Nomura, A.; Sugiura, Y. Inorg. Chem. 2004, 43, 1708-1713.
(7) (a) Laity, J. H.; Lee, B. M.; Wright, P. E. Curr. Opin. Struct. Biol. 2001,
11, 39-46. (b) Wolfe, S. A.; Nekludova, L.; Pabo, C. O. Annu. ReV.
Biophys. Biomol. Struct. 2000, 29, 183-212.
with Kd ) 85 nM.17 As expected, Zn‚2 showed an enhanced
cleavage activity for pUC19GC (Figure 3). Moreover, in sharp
contrast to that of Zn‚1, the reactivity of Zn‚2 for pUC19GC
increased as the ionic strength increases. At [NaCl] ) 200 mM,
Zn‚2 converted pUC19GC even to form III. On the other hand,
Zn‚2 showed a small hydrolytic activity for pUC19 at a high ionic
strength. These results indicate that nonselective binding to DNA
may be prevented at a higher ionic strength, and as a consequence,
the hydrolytic cleavage of the GC box by Zn‚2 was achieved
through specific interactions, such as hydrogen bonding with DNA
bases of the GC box, like the wild-type Sp1.
To specify in detail the cleaved sites within the GC box, a 37
bp DNA duplex containing a GC box was hydrolyzed by Zn‚2.
Figure 4 shows the cleavage sites in the G- and C-strands of the
DNA oligomer, in which the hydrolyzed sites appear as dark bands.
Similar to that in pUC19GC, a higher ionic strength afforded a
higher cleavage activity for the DNA oligomer. In addition, the
results revealed that Zn‚2 hydrolyzes the DNA phosphates around
G(7) on the G-strand and around C(3′), C(6′), and C(7′) on the
C-strand. Cleavage of the two strands at the same positions is
consistent with the transformation of the supercoiled pUC19GC to
form III. Guanine methylation interference studies showed that the
guanine at the 7-position weakly interacts with Zn‚2.17 Such a weak
interaction may cause an effective hydrolytic reaction at around
the G(7) site by Zn‚2.
(8) Kamiuchi, T.; Abe, E.; Imanishi, M.; Kaji, T.; Nagaoka, M.; Sugiura, Y.
Biochemistry 1998, 37, 13827-13834.
(9) Nagaoka, M.; Doi, Y.; Kuwahara, J.; Sugiura, Y. J. Am. Chem. Soc. 2002,
124, 6526-6527.
(10) (a) Segal, D. J.; Beerli, R. R.; Blancafort, P.; Dreier, B.; Effertz, K.; Huber,
A.; Koksch, B.; Lund, C. V.; Magnenat, L.; Valente, D.; Barbas, C. F.,
III. Biochemistry 2003, 42, 2137-2148. (b) Beerli, R. R.; Barbas, C. F.,
III. Nat. Biotechnol. 2002, 20, 135-141. (c) Segal, D. J.; Barbas, C. F.,
III. Curr. Opin. Biotechnol. 2001, 12, 632-637. (d) Sera, T.; Uranga, C.
Biochemistry 2002, 41, 7074-7081.
(11) (a) Koike, T.; Kimura, E. J. Am. Chem. Soc. 1991, 113, 8935-8941. (b)
Kimura, E.; Kodama, Y.; Koike, T.; Shiro, M. J. Am. Chem. Soc. 1995,
117, 8304-8311. (c) Hettich, R.; Schneider, H.-J. J. Am. Chem. Soc. 1997,
119, 5638-5647. (d) Deck, K. M.; Tseng, T. A.; Burstyn, J. N. Inorg.
Chem. 2002, 41, 669-677.
(12) The product of BNP hydrolysis, 4-nitrophenyl phosphate, was quite slowly
hydrolyzed;18 therefore, only the degradation of BNP proceeded under
our experimental conditions (ref 11a).
(13) (a) Koike, T.; Takamura, M.; Kimura, E. J. Am. Chem. Soc. 1994, 116,
8443-8449. (b) Kimura, E. Acc. Chem. Res. 2001, 34, 171-179. (c) Jang,
B.-B.; Lee, K. P.; Min, D. H.; Suh, J. J. Am. Chem. Soc. 1998, 120,
12008-12016.
(14) (a) Kadonaga, J. T.; Jones, K. A.; Tjian, R. Trends Biochem. Sci. 1986,
11, 20-23. (b) Kriwacki, R. W.; Schultz, S. C.; Steitz, T. A.; Caradonna,
J. P. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9759-9763. (c) Kuwahara,
J.; Yonezawa, A.; Futamura, M.; Sugiura, Y. Biochemistry 1993, 32,
5994-6001.
(15) The present DNA cleavage did not change in anaerobic conditions,
indicating that the possibility of a metal ion-driven oxidative cleavage
process can be ruled out.
(16) Yokono, M.; Saegusa, N.; Matsushita, K.; Sugiura, Y. Biochemistry 1998,
37, 6824-6832.
(17) Hori, Y.; Suzuki, K.; Okuno, Y.; Nagaoka, M.; Futaki, S.; Sugiura, Y. J.
Am. Chem. Soc. 2000, 122, 7648-7653.
(18) Kirby, A. J.; Jencks, W. P. J. Am. Chem. Soc. 1965, 87, 3209-3216.
In conclusion, we successfully demonstrated selective hydrolysis
of the DNA duplex at the GC box by the three-tandem zinc finger
JA045663L
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