Template-directed interference footprinting of protein-phosphate contacts in DNA

Article abstract of DOI:10.1021/ol006792j

(equation presented) We have developed a method for interference footprinting of contacted phosphates in protein-DNA complexes. Template-directed enzymatic polymerization using a synthetic triphosphate analogue (αMe-dTTP) generates a product having a modified internucleotide linkage, which perturbs protein-phosphate contacts. We found that treatment of the methylphosphonodiester-substituted extension product under nonaqueous conditions (MeO^-/MeOH) led to the formation of a single cleavage product at each T residue but to two cleavage products when treated under the standard aqueous piperidine cleavage protocol.

Full text of DOI:10.1021/ol006792j

ORGANIC
LETTERS
2001
Vol. 3, No. 1
71-74
Template-Directed Interference
Footprinting of Protein−Phosphate
Contacts in DNA
Orige`ne Nyanguile† and Gregory L. Verdine*
Department of Chemistry and Chemical Biology, HarVard UniVersity,
12 Oxford Street, Cambridge, Massachusetts 02138
Verdine@chemistry.harVard.edu
Received October 27, 2000
ABSTRACT
We have developed a method for interference footprinting of contacted phosphates in protein−DNA complexes. Template-directed enzymatic
polymerization using a synthetic triphosphate analogue (rMe-dTTP) generates a product having a modified internucleotide linkage, which
perturbs protein−phosphate contacts. We found that treatment of the methylphosphonodiester-substituted extension product under nonaqueous
conditions (MeO-/MeOH) led to the formation of a single cleavage product at each T residue but to two cleavage products when treated under
the standard aqueous piperidine cleavage protocol.
Proteins contact DNA through a combination of interactions
with the heterocyclic bases and the deoxyribose-phosphate
backbone.1 Although protein-phosphate contacts do not
generally appear to play a significant role in DNA sequence
discrimination, they often make a dominant contribution to
the overall energetics of protein-DNA recognition.2 High-
resolution structures of protein-DNA complexes reveal not
only that phosphate contacts are extensive3 but also that their
pattern is a highly characteristic feature of each complex.
Determination of electrostatic interactions with the DNA
backbone can therefore yield valuable insights into the
structural and energetic character of protein-DNA inter-
faces; this provides impetus for the development of
methods that enable routine phosphate contact analysis. Here
we report a chemically based method for interference
footprinting of contacted phosphates in protein-DNA
complexes.
The most widely employed technique for mapping contact
phosphates, ethylation interference footprinting, relies on
treatment of DNA with a chemical reagent (ethylnitrosourea)
that ethylates the anionic phosphodiester oxygens.4 The
resulting ethylphosphotriesters are less able than the corre-
sponding phosphates to participate in hydrogen bonding and
Coulombic interactions with a protein; hence they interfere
† Present address: Genetic Therapy, Inc., 9 West Watkins Mill Road,
Gaithersburg, MD 20878.
(1) (a) Larson, C. J.; Verdine, G. L. The Chemistry of Protein-DNA
Interactions. Invited Chapter in Bioorganic Chemistry: Nucleic Acids; Hecht,
S. M., Ed.; Oxford University Press: 1996; pp 324-346. (b) Pabo, C. O.;
Sauer, R. T. Annu. ReV. Biochem. 1992, 61, 1053-1095.
(2) Record, M. T., Jr.; Lohman, T. M.; deHaseth, P. L. J. Mol. Biol.
1976, 107, 145-158.
(3) Mandel-Gutfreund, Y.; Schueler, O.; Margalit, H. J. Mol. Biol. 1995,
253, 370-382.
(4) Siebenlist, U.; Gilbert, W. Proc. Natl. Acad. Sci. U.S.A. 1980, 77,
122-126.
10.1021/ol006792j CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/19/2000

with binding of the protein when present at a position that
is contacted. Although this method has proven valuable in
certain instances, its drawbacks have discouraged routine use.
Among the practical difficulties are capriciousness in the
extent and selectivity of ethylation and the requirement of
handling ethylnitrosourea, a toxic, mutagenic, and potentially
explosive reagent. More fundamental concerns regarding
interpretation originate from the fact that ethyl phosphotri-
esters are generated as a diastereomeric mixture and are a
sterically unconservative replacement for a phosphodiester.
We reasoned that the foregoing limitations might be
overcome by designing an alternative phosphodiester ana-
logue that would (i) obviate the need to use dangerous
reagents by being incorporated into DNA enzymatically, (ii)
exist as a single stereoisomer, (iii) resemble a phosphodiester
sterically but differ electrostatically. Finally (iv) the analogue
should undergo selective chemical scission leading to the
production of a single detectable DNA cleavage product. The
method we have developed to fulfill these criteria is an
extension of template-directed interference (TDI) footprint-
ing, a design-based approach for determining base contacts
in protein-DNA complexes.5
phosphonodiesters can interfere with phosphate contacts,8 and
other data clearly indicate that this linkage is labile toward
aqueous base.9
To test TDI-p footprinting,10 we generated a 183 bp single-
stranded DNA template containing the consensus binding
site for the nuclear factor of activated T cells (NFAT), a
protein that acts as a master regulator of T cell proliferation.11
This template was annealed to a radiolabeled primer, from
which extension was catalyzed by HIV reverse transcriptase
in the presence of RMe-dTTP and the four naturally
occurring 2′-deoxynucleotide 5′-triphosphates (dNTPs). High-
resolution gel electrophoretic analysis of the extension
mixtures and the products of their cleavage with 1 M aqueous
piperidine revealed that an RMe-dNTP:dTTP ratio of 4:1
afforded complete extension, with roughly one methylphos-
phonodiester substitution per DNA molecule, randomly
distributed throughout the DNA pool. However, we found
that each T position in the sequence was represented by two
cleavage products (Figure 2A), which presumably results
from DNA backbone scission on either side of the phos-
phonodiester (cleavage of either bond a or b in Figure 2B);
such product mixtures have the highly undesirable effects
of complicating the interference analysis and decreasing its
sensitivity.12
The key chemical components of our system (TDI-p
footprinting) are 2′-deoxynucleoside 5′-triphosphate ana-
logues having one of the two nonbridging R-phosphate
oxygen atoms replaced by a methyl group (for example,
RMe-dTTP, Figure 1). Enzymatic DNA polymerization using
Noting a report that a simple dinucleoside methylphos-
phonodiester appeared to undergo tandem displacement (at
both bonds a and b, Figure 2C) when treated with MeO-/
MeOH,13 we treated the extension product with 0.5 M
NaOMe in anhydrous MeOH and now observed a single band
at each T position (Figure 2A). We believe that this modified
cleavage procedure will be generally applicable to other labile
(7) Terminal deoxynucleotidyl transferase has been shown to incorporate
only the Sp diastereomer of the methylphosphonodiester into DNA,6a from
which it can be inferred that the enzyme utilizes only the Rp diastereomer
of RMe-dTTP. The stereochemical specificity of terminal deoxynucleotidyl
transferase for analogues having modifications at the R-phosphorus is like
that of all DNA polymerases tested thus far (Lesnikowski, Z. J. Bioorg.
Chem. 1993, 21, 127-155). Although the stereochemical specificity of HIV
reverse transcriptase (RT) for R-modified triphosphate analogues has not
been reported, the recently determined X-ray crystal structure of an RT/
primer-template/nucleotide complex (Huang, H.; Chopra, R.; Verdine, G.
L.; Harrison, S. C. Science 1998, 282, 1669-1675) shows that the enzyme
binds the nucleotide in a way that is nearly identical to that of polymerases
that have been analyzed stereochemically.
Figure 1. Schematic representation of the analogue incorporation
step of TDI-p footprinting. RMe-dTTP is the synthetic triphosphate
analogue used as substrate for template-directed enzymatic poly-
merization. The atoms highlighted in bold are incorporated stereo-
specifically into DNA resulting in the formation of a methyl-
phosphonodiester linkage.
(8) (a) Smith, S. A.; McLaughlin, L. W. Biochemistry 1997, 36,
6046-6058. (b) Pritchard, C. E.; Grasby, J. A.; Hamy, F.; Zacharek, A.
M.; Singh, M.; Karn, J.; Gait, M. J. Nucleic Acids Res. 1994, 22, 2592-
2600. (c) Botfield, M. C.; Weiss, M. A. Biochemistry 1994, 33, 2349-
2355.
(9) Sinha, N. D.; Grossbruchhaus, V.; Ko¨ster, H. Tetrahedron Lett. 1983,
24, 877-880.
(10) A scheme depicting the overall procedure for TDI-p footprinting,
accompanied by a detailed experimental procedure, is available in the
Supporting Information or can be obtained via the Internet at the URL http://
glviris.harvard.edu.
(11) (a) Crabtree, G. R.; Clipstone, N. A. Annu. ReV. Biochem. 1994,
63, 1045-1083. (b) Rao, A.; Luo, C.; Hogan, P. G. Annu. ReV. Immunol.
1997, 15, 707-747.
RMe-dTTP as a substrate6 generates the Sp-configurated7
methylphosphonodiester internucleotide linkage (Figure 1)
on the 5′-side of T residues. Experiments using synthetic,
monosubstituted oligonucleotides indicated that methyl-
(5) (a) Hayashibara, K. C.; Verdine, G. L. J. Am. Chem. Soc. 1991, 113,
5104-5106. (b) Hayashibara, K. C.; Verdine, G. L. Biochemistry 1992,
31, 11265-11273. (c) Mascaren˜as, J. L.; Hayashibara, K. C.; Verdine, G.
L. J. Am. Chem. Soc. 1993, 115, 373-374. (d) Min, C. H.; Cushing, T. D.;
Verdine, G. L. J. Am. Chem. Soc. 1996, 118, 6116-6120.
(6) (a) Higuchi, H.; Endo, T.; Kaji, A. Biochemistry 1990, 29, 8747-
8753. (b) Victorova, L. S.; Dyatkina, N. B.; Mozzherin, D.; Atrazhev, A.
M.; Krayevsky, A. A.; Kukhanova, M. K. Nucleic Acids Res 1992, 20, 783-
789.
(12) Ethylation interference footprints also show mixtures of products
resulting from 5′- and 3′-cleavage of the ethylphosphotriester by hydroxide
ion. The major cleavage pathway, however, involves cleavage of the ethyl
substituent (with liberation of ethanol), a process that regenerates the original
phosphodiester. The dominance with which this cleavage pathway operates
greatly reduces the sensitivity of ethylation interference footprints by erasing
most of the information-bearing adducts.
(13) Kuijpers, W. H.; Kuyl-Yeheskiely, E.; van Boom, J. H.; van Boeckel,
C. A. Nucleic Acids Res. 1993, 21, 3493-3500.
72
Org. Lett., Vol. 3, No. 1, 2001

Figure 2. Products of cleavage of methylphosphonodiester linkages in DNA under aqueous (1 M aqueous piperidine) versus nonaqueous
(0.5 M NaOMe/in anhydrous MeOH) conditions. (A) High-resolution polyacrylamide gel electrophoresis analysis of 5′-radiolabeled DNA
bearing statistically distributed methylphosphonate linkages cleaved under either aqueous (piperidine) or nonaqueous (sodium methoxide)
conditions. The uncleaved control lane is blank, except for minor premature termination products, because the full-length extension product
migrates in the upper position of the gel, which is not shown. Authentic Sanger dideoxy sequencing lanes are shown on the left. Note the
1:1 correspondence between the T Sanger lane and the bands in the base-cleaved DNA samples on the right; the latter migrate somewhat
faster (lower in the gel) because they possess one fewer nucleotide unit. (B) Reaction pathway under aqueous conditions, generating two
methylphosphomonoester products, which migrate differently on an electrophoretic gel. Circled asterisk denotes the location of the 5′-32P
label; only those products that contain this label are detected by autoradiography. (C) Reaction pathway under nonaqueous conditions. The
two methylphosphonodiester products react further to give a single cleaved DNA species.
backbone modifications, such as phosphotriesters and S-
alkylphosphorothioates, greatly improving methods based
thereon.4,14
Having established efficient cleavage conditions, we then
incubated the pool of randomly phosphonodiester-modified
DNA with varying concentrations of human NFATp15 and
separated protein-bound from unbound fractions using native
gel electrophoresis (refer to Supporting Information). Fol-
lowing recovery of the two fractions from the gel, we treated
(15) McCaffrey, P. G.; Luo, C.; Kerppola, T. K.; Jain, J.; Badalian, T.
M.; Ho, A. M.; Burgeon, E.; Lane, W. S.; Lambert, J. N.; Curran, T.;
Verdine, G. L.; Rao, A.; Hogan, P. G. Science 1993, 262, 750-754.
(14) Gish, G.; Eckstein, F. Science 1988, 240, 1520-1522.
Org. Lett., Vol. 3, No. 1, 2001
73

In a TDI footprint,5 interference is evident as those bands
that are weak or missing in the bound fractions while being
correspondingly enriched in the unbound fractions (enrich-
ment is most clearly evident at high protein concentrations,
when most of the DNA molecules containing noninterfering
substitutions have been shifted to the bound fraction). On
the basis of these criteria, we observe interference of NFATp
binding at four phosphates in the noncoding strand of the
consensus site, namely those 5′ to the four T residues;
interference was not observed at any other T position.
Inspection of the solution structure of human NFATc (an
NFATp isoform) bound to DNA17 reveals that the protein
contacts these four phosphates and no others on the non-
coding strand. Closer inspection of these contacts reveals
that interference of NFATp binding caused by methyl
phosphonodiester substitution occurs regardless of whether
the protein more closely approaches the pro-R or pro-S
phosphate oxyanion.
Here we have reported a new design-based method
suitable for the routine analysis of phosphate contacts in
protein-DNA complexes. Although the example shown
applies only to phosphate contacts 5′ to thymine, straight-
forward extension of this procedure using the three other
RMe-dNTPs18 should enable mapping of all such backbone
interactions.
Acknowledgment. We thank Huifang Huang for provid-
ing HIV RT, Cheryl Vaughan for providing NFATp, and
Milan Chytil for experimental advice. We are grateful to
Boehringer-Mannheim Biochemicals and Variagenics, Inc.
for supporting this work.
Supporting Information Available: Detailed descrip-
tion of experimental procedures and 2 figures describing
(i) the TDI-p phosphate footprinting procedure and (ii)
native PAGE separation of protein-bound and unbound
fractions. This material is available free of charge via the
Internet at http://pubs.acs.org.
Figure 3. TDI-p footprinting of the NFATp-mARRE2 complex.
The sequence of the noncoding strand of the consensus NFATp
recognition site16 is shown alongside the gel. The four lanes at the
far left (A, T, G, and C) are an authentic Sanger (dideoxy) sequence
of the template. The remainder of the gel shows T-cleavage ladders
generated after NaOMe/MeOH treatment. Lanes 1-5 represent the
cleavage reaction of the respective bound and unbound fraction
after incubation with NFATp and native gel electrophoresis (see
Supporting Information): lane 1, 2.5 × 10-8 M NFATp; lane 2, 5
Concentration of DNA: 5 × 10-9 M. A bracket indicates positions
at which interference is evident. The two lanes denoted TMe are
duplicate controls in which the extended template was cleaved
without incubation with NFATp.
OL006792J
× 10-8; lane 3, 7.5 × 10-8; lane 4, 1 × 10-7; lane 5, 1.5 × 10-7
.
(16) Chen, L.; Harrison, S. C. Nature 1998, 392, 42-48.
(17) (a) Zhou, P.; Sun, L. J.; Dotsch, V.; Wagner, G.; Verdine, G. L.
Cell 1998, 92, 687-696. (b) NFATc and NFATp possess identical phosphate
contact residues. No high-resolution structure is available for the binary
DNA/NFATp complex, but a ternary complex containing DNA, NFATp,
and AP-1 has been determined.16
(18) (a) Dineva, M. A.; Petkov, D. D. Nucleosides Nucleotides 1996,
15, 1459-1467. (b) Dineva, M. A.; Ivanoc, I. G.; Petkov, D. D. Nucleosides
Nucleotides 1997, 16, 1875-1882.
them in parallel with NaOMe/MeOH and resolved the
cleavage products on a DNA-sequencing gel (Figure 3).
74
Org. Lett., Vol. 3, No. 1, 2001