Double-Strand Hydrolysis of Plasmid DNA
J. Am. Chem. Soc., Vol. 123, No. 9, 2001 1899
H2O, adjusted to pH 7, and sterile filtered prior to use. The reactions
were carried out at 37 and 55 °C in a covered heating block to prevent
solvent evaporation in the course of the experiment. Samples were
frozen at -20 °C in a dye solution (0.04% bromophenol blue, 0.04%
xylene cyanol FF, and 5% glycerol) prior to running the gel. All gels
were run on 1.2% agarose slab gels for 120 min at 70 V. Gels were
stained with ethidium bromide and pictures taken with Polaroid 3000ISO
type 667 film. Bands on the gels were quantified by using a Microtek
Scanmaker E6 and the software program NIH Image 1.60. This
quantification was confirmed by directly digitizing the gel image with
use of a Biorad GelDoc and the software Molecular Analyst. The
amount of supercoiled DNA was multiplied by a factor of 1.22 to
account for reduced ethidium bromide intercalation into supercoiled
DNA.
Figure 1. Structures of the HXTA, HXTP, and HPTA ligands.
The values for n1 and n2 were determined from the aforementioned
gel quantification and formulas based on the Freifelder-Trumbo
relationship.24 The amount of supercoiled DNA is expressed in fI )
exp(-(n1 + n2)), where fI is the fraction of supercoiled DNA and n1
and n2 are the number of single-strand and double-strand DNA cuts,
respectively. The amount of linear DNA is expressed in fIII ) n2
exp(-n2), where fIII is the fraction of DNA in the linear form. The n1
and n2 values reported in this paper represent 42 data points taken from
9 gels for Ce2(HXTA). Alternatively, this method of analysis was
confirmed by using Cowan’s methodology.25 In this method n2 ) fIII/
(1 - fIII) and n1 ) -ln(fI(1 + n2)) are the formulas used and are specific
for cases where double-strand cuts are the dominant mechanism for
generating linear DNA.
the possible double-strand hydrolysis reaction has not been
reported.22 Here we follow up on our earlier report of dicerium
and dilanthanide complexes that catalyze double-strand DNA
hydrolysis at 55 °C17 with a detailed study of a new dicerium
complex Ce2(HXTA) (for the structure of HXTA, see Figure
1), which is capable of carrying out double-strand DNA
hydrolysis efficiently at 37 °C. Furthermore, Ce2(HXTA)
exhibits high regioselectivity in cleaving the 3′-O-P bond.
Materials and Methods
Kinetics. Kinetic determinations on the cleavage of supercoiled
plasmid DNA by Ce2(HXTA) were carried out by quantifying the
nicked and linear DNA fractions as described above. Plots of ln(nicked
fraction) or ln(linear fraction) values versus time afforded a straight
line, the slope of which provides the apparent first-order rate constant.
Kinetic measurements with bis(p-nitrophenyl)phosphate (BNPP) were
performed on a Beckman DU-650 spectrophotometer at 37 °C in 100
mM pH 8.0 Tris buffer. Reactions with 0.1 mM Ce2(HXTA) and 1
mM dApdA were performed at 37 °C for 24 h in 10 mM pH 8.0 Tris
buffer. At the end of the reaction, Ce2(HXTA) was precipitated with 1
M KH2PO4. The supernatant was then applied on a C18 reverse phase
column for the analysis of hydrolysis products, using as eluant a 50:50
mixture of MeOH:KH2PO4 at pH 4.5 with a flow rate of 0.5 mL/min.
The Ce2(HXTA) reaction products were confirmed via known standards.
Substances. The Litmus 29 plasmid DNA, prepared from DH5R
cells, was purified by using Qiagen Plasmid Maxi Kits. The enzymes
Hind III, PVu II, calf intestinal phosphatase, and RQ DNase I were
purchased from Promega, BamH I and T4 polynucleotide kinase were
purchased from New England Biolabs, and the Klenow fragment of
DNA polymerase I was purchased from Gibco BRL. MPE was
purchased from Sigma. All radioactive nucleotides were ordered from
Amersham Life Sciences. Metal salts were purchased from Aldrich.
All materials used were molecular biology grade when available,
otherwise the purest available material was used.
The ligand HXTA was synthesized by a modification of published
procedures.23 To a 100 mL aqueous solution of 16.7 g (0.125 mol) of
iminodiacetic acid and 6.8 mL (6.75 g, 0.063mol) of p-cresol was added
10.5 g (0.25 mol) of NaOH in 40 mL of water cooled in an ice-water
bath. Upon dissolution, 15 mL of 37% formaldehyde was added
dropwise at 0 °C. The solution was stirred for 30 min, heated at 70 °C
for 4 h, and then concentrated to dryness. Recrystallization of the solid
from methanol yielded colorless crystals of Na4HXTA. Yield ∼90%.
1H NMR in D2O (δ, ppm): 6.83 (s, 2H), 3.60 (s, 4H), 3.04 (s, 8H),
and 2.05 (s, 3H). Elemental analysis (calculated) for C17H18N2O9Na4‚
1.7H2O: C 39.52 (39.49), H 4.44 (4.17), N 5.28 (5.41). The HXTP
ligand was also synthesized as the tetrasodium salt. Six milliliters of a
37% formaldehyde solution (80 mmol) was added in a dropwise fashion
to a 50 mL aqueous solution of disodium iminodipropionate (10 g, 48
mmol) and p-cresol (2.5 mL, 24 mmol) at 0 °C. This mixture was kept
at 0 °C for 30 min and then stirred at room temperature for 48 h. The
solvent was removed by rotary evaporation, and the resulting residue
was recrystallized from methanol (15.6 g, 60% yield). 1H NMR in D2O
(δ, ppm): 6.85 (s, 2H), 3.63 (s, 4H), 2.73 (t, 8H, JCH,CH ) 7.8 Hz),
2.30 (t, 8H, JCH,CH ) 7.8 Hz), 2.06 (s, 3H). Elemental analysis
(calculated) for C21H26N2O9Na4‚3.6H2O: C 41.56 (41.54), H 5.37 (5.51),
N 4.54 (4.61).
End Analysis of Restriction Fragments. 5′-end labeled restriction
fragments were prepared by using the following procedure. Litmus 29
plasmid DNA was treated with 50 U of Hind III followed by ethanol
precipitation. The DNA was then treated with 3 U of calf intestinal
phosphatase followed by heat treatment at 75 °C for 10 min in the
presence of 10 mM pH 8 EDTA. After standard phenol:chloroform:
isoamyl alcohol treatment and ethanol precipitation the DNA was 5′-
end-labeled with 2 U T4 polynucleotide kinase in the presence of γ-32P-
ATP. The DNA was then treated with phenol:chloroform:isoamyl
alcohol and run through a Sephadex G-50 column equilibrated to pH
8 with Tris buffer. After NH4OAc/ethanol precipitation, the DNA was
treated with 4 U of PVu II and purified with a 6% nondenaturing PAGE
gel. The DNA was visualized by exposure to X-ray film and purified
from the gel by using the crush and soak method.26
3′-end labeled restriction fragments were prepared using the fol-
lowing procedure. Litmus 29 DNA (2 µg) was treated with 15 U of
BamH I at 37 °C followed by phenol:chloroform:isoamyl alcohol
extraction and ethanol precipitation. The DNA was then treated with 4
U of the Klenow fragment and R-32P-dATP on ice with use of standard
fill-in conditions, provided by Gibco BRL, and extracted with phenol:
chloroform:isoamyl alcohol followed by a Sephadex G-50 spin column
and ethanol precipitation. The sample was then treated with 20 U of
PVu II at 37 °C and purified on a 6% nondenaturing PAGE gel. The
Plasmid DNA Cleavage. The plasmid DNA cleavage reactions were
carried out in a total reaction volume of 70 µL containing cleavage
agent, 7 µg of Litmus 29 plasmid DNA (2820 base pairs), and 7 µL of
100 mM pH 8.0 Tris buffer. La(NO3)3, Ce(NO3)3, Nd(NO3)3, Eu(NO3)3,
Yb(NO3)3, Fe(NO3)3, Zn(NO3)2, (NH4)2Fe(SO4)2, and (NH4)2Ce(NO3)6
were the sources of the metal ions used in the cleavage reactions.
Solutions (0.1 M) of HXTA and HXTP were made with sterile Millipore
(24) Freifelder, D.; Trumbo, B. Biopolymers 1969, 7, 681-693.
(25) Cowan, R.; Collis, C. M.; Grigg, G. W. J. Theor. Biol. 1987, 127,
229-245.
(26) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning. A
Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, 1989.
(22) Fitzsimons, M. P.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 3379-
3380.
(23) Murch, B. P.; Bradley, F. C.; Boyle, P. D.; Papaefthymiou, V.; Que,
L., Jr. J. Am. Chem. Soc. 1987, 109, 7993-8003.