Dda helicase unwinds a DNA-PNA chimeric substrate: Evidence for an inchworm mechanism

Article abstract of DOI:10.1016/j.bmcl.2006.01.013

Helicases are ubiquitous enzymes involved in all aspects of DNA metabolism including replication, repair, recombination, and transcription. The mechanism of the bacteriophage T4 Dda helicase was investigated by preparing a DNA-PNA chimeric substrate. Surp

Full text of DOI:10.1016/j.bmcl.2006.01.013

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1816–1820
Dda helicase unwinds a DNA–PNA chimeric substrate:
Evidence for an inchworm mechanism
Travis L. Spurling, Robert L. Eoff and Kevin D. Raney*
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
Received 12 November 2005; revised 2 January 2006; accepted 5 January 2006
Available online 24 January 2006
Abstract—Helicases are ubiquitous enzymes involved in all aspects of DNA metabolism including replication, repair, recombina-
tion, and transcription. The mechanism of the bacteriophage T4 Dda helicase was investigated by preparing a DNA–PNA chimeric
substrate. Surprisingly, Dda was able to unwind a substrate containing 12 PNA moieties in the loading strand of the enzyme. We
suggest a mechanism whereby the Dda helicase contains two distinct DNA binding domains which allow an inchworm mechanism
for translocation. A single step of the enzyme is sufficient to unwind the DNA–PNA chimera because several base pairs melt spon-
taneously due to thermal fraying. Hence, Dda helicase can unwind the substrate without actually translocating along the PNA.
Ó 2006 Elsevier Ltd. All rights reserved.
Helicases are enzymes that unwind duplex DNA to pro-
vide ssDNA intermediates during DNA replication,
recombination, and repair.1,2 An inchworm mechanism
has been suggested for some helicases whereby two
DNA binding sites are contained on the enzyme.3,4
The inchworm mechanism requires that one binding site
maintain contact with the DNA while the other binding
site moves along the lattice in a process driven by ATP
binding and hydrolysis. The mechanism of Dda helicase
from Bacteriophage T4 was investigated in this study.
Dda can function as a monomeric helicase during
unwinding of short oligonucleotide substrates, suggest-
ing that an inchworm mechanism might apply to this
enzyme.5,6
PNA, forming a DNA–PNA hybrid. Standard PNAs
are achiral, electrostatically neutral, chemically and bio-
logically stable, and synthetically compatible with stan-
dard solid-phase chemistry protocols.7,8 PNAs have
demonstrated sequence-specific, efficient in vitro inhibi-
tion of telomerase activity,9 HIV replication,10 bacteria
multiplication,11 ribosomal RNA function,12 eukaryotic
RNA translation,13 and DNA polymerase activity.14
Dda helicase was able to unwind the DNA–PNA hybrid
substrate at rates that were similar to that of a normal
DNA substrate.15 Subsequent studies indicated that
Dda does not bind tightly to PNA.16 These results
support a mechanism whereby Dda unwinds the
DNA–PNA hybrid substrate by translocating along the
loading strand of DNA and stripping away the comple-
mentary PNA strand through steric interactions that
force apart the duplex; that is, a snowplow or wirestrip-
per mechanism.17,18 More recently, a DNA–PNA–DNA
chimera was prepared as a substrate for Dda.19 Mono-
meric Dda was unable to unwind this substrate, indicat-
ing that a single PNA moiety in the loading strand is able
to block the monomeric form of the helicase.
In vitro, most helicases must initiate unwinding by first
binding to a region of ssDNA. These enzymes translo-
cate unidirectionally into the duplex region resulting in
separation of the two strands. The strand on which the
helicase translocates is referred to as the loading strand,
whereas the complementary strand is called the displaced
strand. The mechanism for unwinding by Dda was previ-
ously investigated by preparing a substrate in which the
displaced DNA strand was substituted with a strand of
In this report, a DNA–PNA chimera has been utilized as
a helicase substrate. We created a substrate that con-
tains 8 nt of ssDNA that serves as a loading site for
the helicase. Adjacent to the loading site are 12 PNA
moieties which were hybridized to a complementary
12mer of ssDNA to complete the substrate (Fig. 1).
We have previously shown that a normal DNA sub-
strate containing 12 bp is readily unwound by Dda.6
Abbreviations: MMT, monomethoxytrityl; DMT, dimethoxytrityl;
PNA, peptide nucleic acid; ss, single stranded; ds, double stranded.
Keywords: Helicase; Peptide nucleic acids; PNA; DNA–PNA chimera;
Unwinding.
*
Corresponding author. Tel.: +1 501 686 5244; e-mail: raneykevind@
uams.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.013

T. L. Spurling et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1816–1820
1817
Figure 1. Substrates designed for investigating the mechanism of Dda
helicase. (A) Schematic illustration of the DNA–PNA chimera
(uppercase letters = PNA; lowercase letters = DNA). The structure of
the PNA linker and a PNA moiety are shown at the interface with the
DNA sequence. (B) A substrate for the helicase was prepared by
hybridizing the chimera to a complementary strand of DNA to form
the chimera:DNA substrate. A well-characterized DNA substrate
served as a positive control and a DNA–PNA hybrid was examined to
compare the PNA location in the loading strand or in the displaced
strand of the helicase substrate.
Scheme 1. (A) MMT-protected aminoethyl PNA glycine backbone
synthesis. (B) DMT-protected hydroxyl ethyl PNA–DNA linker
backbone synthesis.
(4) in moderate yield (41%) for two steps overall
(Scheme 1A).
A PNA–DNA linker has also been developed to connect
DNA to a PNA strand.21 In place of the standard ami-
noethyl glycine backbone, the PNA–DNA linker back-
bone possesses a hydroxyethyl glycine unit. Synthesis
of the PNA–DNA linker backbone requires a third step
but only two pots, and the reaction time is only slightly
longer. Dimethoxytrityl chloride was added to ethylene
glycol as solvent (Scheme 1B). After 30 min, the protect-
ed ethylene glycol (5) was extracted with ethyl acetate/
water. Mesyl chloride with triethylamine was used to
convert the alcohol to a good leaving group in less than
an hour followed by removal of solvent and addition of
triethylamine and methyl ester glycine to the same pot.
Refluxing for 3 h gave the desired DMT-protected
hydroxyethyl glycine, PNA–DNA linker backbone. Col-
umn purification gave the pure backbone (6) in good
yield (61%) for three steps. The backbones (4 and 6)
were used to make standard PNA monomers (1) and
the PNA–DNA linker (2) using previously published
procedures.22
Synthesis of PNA monomers. The DNA–PNA chimera
was prepared using PNA monomers containing protect-
ing groups that are compatible with DNA synthesis
(Fig. 2).
Synthesis of the PNA backbone was performed by a
modified two-step reaction sequence.20 The reaction be-
tween ethylenediamine as solvent and monomethoxytri-
tyl chloride proceeded in 30 min to the desired protected
ethylenediamine (Scheme 1A, compound 3). Refluxing
(3) with methyl bromoacetate in methylene chloride
for 2.5 h followed by flash chromatography gave the
desired standard aminoethyl glycine PNA backbone
Solid-phase synthesis of the DNA–PNA chimeric mole-
cules. The chimeric sequence was designed to serve as
mechanistic probe for DNA unwinding by the bacterio-
phage T4 helicase, Dda. Dda (DNA dependent ATPase)
is classified as a superfamily I helicase that has a 50 to 30
directional bias for unwinding. Dda binds to the
Figure 2. PNA monomers (1), PNA–DNA linker monomer (2).

1818
T. L. Spurling et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1816–1820
50-ssDNA region of the substrate and then translocates
into the duplex. We have previously prepared PNA
strands by manual synthesis.23 However, numerous at-
tempts to manually prepare the 20mer DNA–PNA chi-
mera failed. The successful synthesis of the chimera was
performed on a modified Expedite 8909 DNA Synthe-
sizer (see Supplementary materials for details). Follow-
ing synthesis, the chimera was cleaved from the resin
and deprotected by treatment with concentrated
anhydrous methanolic ammonia. Size-exclusion chro-
matography and denaturing polyacrylamide gel electro-
phoresis were then used to remove any truncated
species. The identity of the chimera was confirmed by
MALDI-TOF mass spectrometry (predicted mass =
5736.7 g/mol; measured m/z = 5737.8 [M+Na+H]1+).
Dda helicase-catalyzed unwinding of the DNA–PNA chi-
mera. The 20mer chimeric sequence was designed to
allow an eight base ssDNA overhang for binding to
the helicase and a 12 base duplex after hybridization
with a 12 nt complementary strand. The question of
whether unwinding could occur when the enzyme
encountered the PNA was examined. Three different
substrates were compared for DNA unwinding by Dda
helicase. In addition to the aforementioned
DNA–PNA chimera,
a well-characterized, normal
DNA substrate was chosen as a positive control
(Fig. 1B). The positive control substrate contains a 15
nt ssDNA overhang and 15 bp, and was previously
shown to serve as an excellent substrate for Dda heli-
case.15 A DNA–PNA hybrid substrate was also exam-
ined in which the loading strand contains only DNA
while the complementary strand contains PNA
(Fig. 1B). For each experiment, the substrate (100 nM)
was incubated in unwinding buffer with Dda helicase
(1 nM) under steady state conditions (excess substrate).
These conditions strongly favor binding of one Dda
molecule per substrate. As expected, the normal DNA
duplex was readily unwound by the helicase (Fig. 3D,
diamonds). We previously reported that the
DNA–PNA hybrid serves as a substrate for Dda heli-
case under conditions in which the enzyme is in excess
of the substrate.15 Excess enzyme conditions favor bind-
ing of more than one Dda helicase molecule to the sub-
strate. When substrate is in excess, only one molecule of
Dda helicase binds to the substrate, but when the en-
zyme is in excess, multiple molecules of Dda can bind
to the substrate depending on the length of the ssDNA
overhang.19,24 In Figure 3D, we show that the same
DNA–PNA hybrid also serves as a substrate under con-
ditions in which the substrate is in great excess over the
enzyme, favoring the monomeric form of Dda helicase
(Fig. 3D, open squares). Finally, the DNA–PNA chime-
ra was found to serve as a substrate for DNA unwinding
by Dda helicase (Fig. 3D, filled circles). The rate of
unwinding of the DNA–PNA chimera substrate was
very similar to that of the normal DNA substrate and
the DNA–PNA hybrid substrate. In light of the pre-
vious results that show little or no affinity between
Dda helicase and PNA,16 and previous results indicating
that monomeric Dda cannot translocate passed a single
PNA moiety,19 unwinding of the DNA–PNA chimera is
most surprising.
Figure 3. Helicase-catalyzed DNA unwinding. (A) Radiolabeled DNA
substrate is incubated with helicase followed by initiation of the
reaction by addition of ATP, Mg(OAc)2, and DNA trap. The DNA
trap prevents re-annealing of the ssDNA products. One hundred
nanomolar substrate was incubated with 1 nM Dda helicase and
aliquots were quenched at varying times by addition of 400 mM
EDTA. (B) Helicase-catalyzed unwinding was measured by electro-
phoretic separation of ssDNA from dsDNA on a native polyacryl-
amide gel for the chimera substrate (B) or the normal DNA substrate
(C) at increasing times. One lane (heat) in each image was produced by
heating an aliquot of the reaction mixture to 95 °C, followed by slow
cooling to room temperature. (D) Unwinding of the DNA–PNA
chimera in the presence of ATP (filled circles) or the absence of ATP
(filled squares) is shown. For comparison, unwinding of a normal
DNA substrate (filled diamonds) or a DNA–PNA hybrid substrate
(open squares) under identical conditions is shown. The rate of
product formation was determined by fitting the early phase of the
reaction to
a ,
linear function resulting in rates of 0.27 nM sꢀ1
0.34 nM sꢀ1, and 0.33 nM sꢀ1 for the DNA–PNA chimera, normal
DNA substrate, and DNA–PNA hybrid, respectively.

T. L. Spurling et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1816–1820
1819
An explanation for how Dda can unwind the
DNA–PNA chimera can be provided when considering
the proposed mechanisms of unwinding by helicases. A
helicase is thought to unwind duplex DNA by melting a
given number of base pairs per catalytic cycle. Various
mechanisms have been proposed in which a helicase
may unwind one base pair per catalytic cycle25 or more
than one base pair per cycle.26 Regardless of the number
of base pairs unwound per catalytic cycle, longer duplex
DNA substrates will require more catalytic steps than
shorter duplexes. A helicase need not reach the end of
a duplex for DNA unwinding to be observed. Other
groups have recently reported spontaneous melting of
the final 9–11 base pairs of duplex DNA, depending
on the particular helicase being studied.27,28 This means
that as the helicase approaches the end of a duplex, the
final base pairs melt spontaneously, giving rise to
ssDNA products. The DNA–PNA chimera used in this
report is only 12 bp in length. Hence, Dda helicase need
only unwind 1–3 base pairs in order for the remaining
base pairs to melt spontaneously.
aromatic amino acids as has been shown with other heli-
cases.25,29,30 The resulting model suggests that Dda can
overcome a 12-PNA block in the loading strand by melt-
ing bases in one step through interactions between the
enzymes 30-binding domain and the nucleobases of
PNA, while the 50-DNA binding domain remains bound
to the ssDNA portion of the chimera. The remaining bp
then spontaneously separate, consistent with estimates
for this minimal dsDNA value found in other helicase
studies.27,28 A previous substrate containing a single
PNA moiety was not unwound under pre-steady state
conditions. However, this substrate contained 16 bp
compared to the 12 bp in the substrate reported here.19
The 16 bp substrate would not be expected to melt after
unwinding of only 1–3 base pairs. Additionally, our re-
sults provide evidence that Dda interacts with DNA
through at least two distinct DNA binding domains.
The presence of two DNA binding domains and the
non-specific displacement of the complementary PNA
strand support a mechanism in which Dda translocates
in a manner analogous to an inchworm and strips away
the complementary strand. If this model for spontane-
ous melting of the final 8 base pairs applies to other heli-
cases, then reported kinetic step sizes that do not
account for spontaneous melting might overestimate
the actual kinetic step size.
A model invoking an inchworm mechanism for Dda and
spontaneous melting of the final 9–11 base pairs of the
substrate can explain the unwinding of the DNA–PNA
chimera. Dda must bind to the ssDNA loading site in
order to initiate the first step for unwinding (Fig. 4).
The first catalytic step likely requires movement of a
sub-domain of the enzyme along the nucleic acid which
results in melting of one or more base pairs. After the
first 1–3 base pairs are unwound, the remaining bp sep-
arate spontaneously due to thermodynamic fraying.
Therefore, Dda is able to unwind the substrate without
continuously translocating along the PNA.
Acknowledgments
This investigation was supported by National Institutes
of Health Grant R01 GM 059400 (K.D.R.) and by NIH
COBRE Grant P20 RR15569 (F. Millet, University of
Arkansas). We thank Dr. Tom Goodwin and Hendrix
College for use of their NMR spectrometer.
Interactions between Dda and the PNA need be only
transient and could occur through base stacking with
Supplementary material
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2006.
01.013.
References and notes
1. Delagoutte, E.; von Hippel, P. H. Q. Rev. Biophys. 2002,
35, 431.
2. Delagoutte, E.; von Hippel, P. H. Q. Rev. Biophys. 2003,
36, 1.
3. Soultanas, P.; Wigley, D. B. Curr. Opin. Struct. Biol. 2000,
10, 124.
4. Yarranton, G. T.; Gefter, M. L. Proc. Natl. Acad. Sci.
U.S.A. 1979, 76, 1658.
5. Morris, P. D.; Tackett, A. J.; Babb, K.; Nanduri, B.;
Chick, C.; Scott, J.; Raney, K. D. J. Biol. Chem. 2001, 276,
19691.
6. Nanduri, B.; Byrd, A. K.; Eoff, R. L.; Tackett, A. J.;
Raney, K. D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
14722.
7. Nielsen, P. E.; Egholm, M. Curr. Issues Mol. Biol. 1999, 1,
89.
8. Norton, J. C.; Piatyszek, M. A.; Wright, W. E.; Shay, J.
W.; Corey, D. R. Nat. Biotechnol. 1996, 14, 615.
Figure 4. Model for Dda-catalyzed unwinding of the 20:12, chime-
ra:DNA substrate. Dda is shown bound to a 50 DNA–PNA chimera
(where PNA is shown as a bold line). ATP binding leads to a
conformational change of the enzyme at the ss/ds DNA junction in
which the DNA binding domains move relative to each other. ATP
hydrolysis, which is coupled to helicase action, leads to unwinding of 1–3
base pairs of duplex due to transient interaction between Dda and the
bases of the PNA strand. The initial helicase ‘step’ displaces enough base
pairs to allow the remaining base pairs to spontaneously melt apart.

1820
T. L. Spurling et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1816–1820
20. Fader, L. D.; Boyd, M.; Tsantrizos, Y. S. J. Org. Chem.
9. Harrison, J. G.; Frier, C.; Laurant, R.; Dennis, R.; Raney,
K. D.; Balasubramanian, S. Bioorg. Med. Chem. Lett.
1999, 9, 1273.
10. Koppelhus, U.; Zachar, V.; Nielsen, P. E.; Liu, X.;
Eugen-Olsen, J.; Ebbesen, P. Nucleic Acids Res. 1997,
25, 2167.
2001, 66, 3372.
21. Petersen, K. H.; Jensen, D. K.; Egholm, M.; Nielsen, P.
E.; Buchardt, O. Bioorg. Med. Chem. Lett. 1995, 5, 1119.
22. Domling, A.; Chi, K. Z.; Barrere, M. Bioorg. Med. Chem.
Lett. 1999, 9, 2871.
11. Good, L.; Awasthi, S. K.; Dryselius, R.; Larsson, O.;
Nielsen, P. E. Nat. Biotechnol. 2001, 19, 360.
12. Good, L. Cell Mol. Life Sci. 2003, 60, 854.
13. Knudsen, H.; Nielsen, P. E. Nucleic Acids Res. 1996, 24,
494.
14. Taylor, R. W.; Chinnery, P. F.; Turnbull, D. M.;
Lightowlers, R. N. Nat. Genet. 1997, 15, 212.
15. Tackett, A. J.; Morris, P. D.; Dennis, R.; Goodwin, T. E.;
Raney, K. D. Biochemistry 2001, 40, 543.
16. Tackett, A. J.; Corey, D. R.; Raney, K. D. Nucleic Acids
Res. 2002, 30, 950.
23. Goodwin, T. E.; Holland, R. D.; Lay, J. O.; Raney, K. D.
Bioorg. Med. Chem. Lett. 1998, 8, 2231.
24. Byrd, A. K.; Raney, K. D. Nat. Struct. Mol. Biol. 2004,
V11, 531.
25. Velankar, S. S.; Soultanas, P.; Dillingham, M. S.; Subra-
manya, H. S.; Wigley, D. B. Cell 1999, 97, 75.
26. Ali, J. A.; Lohman, T. M. Science 1997, 275, 377.
27. Galletto, R.; Jezewska, M. J.; Bujalowski, W. J. Mol. Biol.
2004, 343, 83.
28. Levin, M. K.; Wang, Y. H.; Patel, S. S. J. Biol. Chem.
2004, 279, 26005.
17. von Hippel, P. H. Nat. Struct. Mol. Biol. 2004, 11, 494.
18. Kawaoka, J.; Jankowsky, E.; Pyle, A. M. Nat. Struct.
Mol. Biol. 2004, 11, 526.
29. Kim, J. W.; Seo, M. Y.; Shelat, A.; Kim, C. S.; Kwon, T.
W.; Lu, H. H.; Moustakas, D. T.; Sun, J.; Han, J. H. J.
Virol. 2003, 77, 571.
19. Eoff, R. L.; Spurling, T. L.; Raney, K. D. Biochemistry
2005, 44, 666.
30. Korolev, S.; Hsieh, J.; Gauss, G. H.; Lohman, T. M.;
Waksman, G. Cell 1997, 90, 635.