B, Roma, Italy;4SISSA, Trieste, Italy The structural changes produced by the minor-groove binding ligand DAPI 4¢,6-diamidine-2-phenylindole on individual strands of trinucleotide repeat
Trang 1Interaction of DAPI with individual strands of trinucleotide repeats
Effects on replication in vitro of the AATÆATT triplet
Edoardo Trotta1, Nicoletta Del Grosso1, Maura Erba2, Sonia Melino2, Daniel Cicero2,3,4and Maurizio Paci2,3
1
Istituto di Neurobiologia e Medicina Molecolare, Consiglio Nazionale delle Ricerche, Roma, Italy;2Dipartimento di Scienze e Tecnologie Chimiche, Universita` di Roma ‘Tor Vergata’, Roma, Italy;3INFM sez B, Roma, Italy;4SISSA, Trieste, Italy
The structural changes produced by the minor-groove
binding ligand DAPI (4¢,6-diamidine-2-phenylindole) on
individual strands of trinucleotide repeat sequences were
detected by electrophoretic band-shift analysis and related to
their effects on DNA replication in vitro Among the 20
possible single-stranded trinucleotide repeats, only the T-rich
strand of the AATÆATT triplet exhibits an observable
fluorescence band and a change in electrophoretic mobility
due to the drug binding This is attributable to the property
of DAPI that favours folding of the random coil ATT strand
into a fast-migrating hairpin structure by a minor-groove
binding mechanism Electrophoretic characteristics of AAT,
ACT, AGT, ATG and ATC are unchanged by DAPI,
suggesting the crucial role of TÆT with respect to AÆA, CÆC and GÆG mismatch, in favouring the binding properties and the structural features of the ATT–DAPI complexes Primer extension experiments, using the Klenow fragment of DNA polymerase I, demonstrate that such a selective structural change at ATT targets presents a marked property to stall DNA replication in vitro in comparison with the comple-mentary AAT and a random GC-rich sequence The results suggest a novel molecular mechanism of action of the DNA minor-groove binding ligand DAPI
Keywords: DAPI; trinucleotide repeats; Klenow; DNA structure; hairpin
DAPI (4¢,6-diamidino-2-phenylindole) is a DNA
minor-groove binding ligand used largely as a fluorescent dye for
DNA and chromosomes [1] It interferes with the activity
of DNA processing enzymes involved in regulatory and
structural functions such as DNA polymerase I [2], RNA
polymerases [2–4], topoisomerases [2,5–7], DNA ligase [2],
exonuclease III [2], DNAase I [8] and restriction
endonuc-leases [8], showing varying levels of inhibitory effects [2]
DAPI preferentially binds into the minor-groove of at least
two consecutive AÆT base-pairs [9,10] or TÆT mismatches
flanked by AÆT base-pairs [11] Quite different binding
mechanisms and a lower affinity with DNA sequences that
contain no adjacent AÆT base-pairs have been reported:
intercalation [12,13], major-groove binding [14] and
p,p-stacking interactions with double-helix ends [15] It has also
been shown that DAPI favours folding into hairpin
structures of the T-rich strand of AATÆATT trinucleotide
repeat sequences [16] In this work we investigate the effects
of DAPI on replication in vitro of AATÆATT trinucleotide
repeats in relation to the structural changes induced by the
drug on individual strands of trinucleotide repeats The
AATÆATT triplet presents a number of biological features
that require further investigation to understand the possible
role of this frequent class of trinucleotide repeat This triplet
has been reported as the most abundant and polymorphic trinucleotide repeat in the human genome [17] Distribution
of the AATÆATT trinucleotide repeat does not appear to
be random in relation to different types of genomic sequences, but it is frequent in introns and uncommon in exons, suggesting a possible role in regulating transcription [18–20] In contrast to the CG-rich triplet repeats associated with human diseases, AATÆATT did not show any pausing
of DNA polymerases in the primer extension assay [21] and has not yet been found associated with human diseases, although its expansion was observed during DNA replica-tion in vitro [22,23] Also unlike other trinucleotide repeats, the AATÆATT repeat in plasmids shows the unusual propensity to adopt nonhydrogen-bonded structures, sug-gesting that it may play a different role in gene regulation [21] The AATÆATT repeat also constitutes a binding site for
at least one nuclear protein [24]
The present study demonstrates that DAPI causes a sequence- and strand-dependent stalling of DNA poly-merase at AATÆATT trinucleotide repeat regions This effect
is associated with the strongly selective property of DAPI to bind and favour the hairpin structure of the T-rich strand of the AATÆATT trinucleotide repeat Our results suggest a novel mechanism of action of DAPI and show the unusual replication feature of a common class of genomic DNA that have unknown functions
Materials and methods
Materials DNA 18-mers: (AAC)6, (AAG)6, (AAT)6, (ACC)6, (ACG)6, (ACT)6, (AGC)6, (AGG)6, (AGT)6, (ATC)6, (ATG), (ATT), (CCG), (CCT), (CGG), (CGT),
Correspondence to E Trotta, Istituto di Neurobiologia e Medicina
Molecolare, Consiglio Nazionale delle Ricerche, Via del Fosso del
Cavaliere 100, 00133 Roma, Italy.
Fax: + 39 064 993 4257, Tel.: + 39 064 993 4567,
E-mail: edoardo.trotta@ims.rm.cnr.it
Abbreviation: DAPI, 4¢,6-diamidino-2-phenylindole.
(Received 5 September 2003, revised 10 October 2003,
accepted 14 October 2003)
Trang 2(CTG)6, (CTT)6, (GGT)6 and (GTT)6 were used for
evaluating electrophoretic band-shifts induced by DAPI
on trinucleotide repeat sequences
Three different 70-base templates for primer extension
experiments were synthesized with a common 40-base
sequence at the 3¢-end constituted by a random region with
no adjacent AÆT bases: 5¢-GGTCCCAGTCCACTCTGT
CGCCGCACCCTGCGGACCTGCT-3¢ (Fig 1A,B) The
remaining 30-base sequence at the 5¢-end was distinctive for
each template: (ATT)10, (AAT)10 and 5¢-CAGGTGGA
GCTGTGTCAGTGCCACTGACCC-3¢ for ATT-,
AAT-and rAAT-andom-template, respectively (Fig 1B) A
5¢-fluoresc-ein labeled 15-base primer was synthesized that was
complementary to the 41–55 template region
DAPI (Fig 1C) and Klenow fragment [3¢ fi 5¢
exonuc-lease-deficient (exo-) of Escherichia coli DNA polymerase I]
were purchased from Sigma (St Louis, MO, USA) and New
England Biolabs Inc (Beverly, MA, USA), respectively
Electrophoretic mobility-shift analysis
DAPI-induced electrophoretic mobility-shift analyses were
performed on 12% (w/v) native polyacrylamide gel at 4C
Before gel inoculation, DNA samples were denatured at
90C and slowly annealed to room temperature The DAPI–DNA complexes were prepared by adding the drug after DNA annealing Polyacrylamide gels were stained with 0.1% (v/v) methylene blue in 0.5Msodium acetate Samples for comparing binding effects of DAPI on the 18-mer trinucleotide repeat sequences were 150 lM DAPI and 35 lM DNA strand in Tris/borate/EDTA buffer Primer–template DNA samples were 50 lM DAPI and
8 lMprimer–template in Tris/borate/EDTA buffer Fluorescence
Fluorescence spectra were recorded on a SPEX FluoroMax photon counting spectrofluorometer equipped with a ther-mostatic cell holder (SPEX Industries Inc., Edison, NJ, USA) Excitation (350 nm) and emission (448 nm) with 1.5 nm band-pass were used and spectra were corrected for background signal The absorbance at the excitation wave-length was less than 0.03 ([DAPI]¼ 1 lM) making the inner filter effects negligible The experiments were per-formed at 37C, in 100 mMNaCl and 10 mMphosphate buffer at pH 7.00, by adding an increasing amount of DNA oligomers to the drug solutions up to 2 : 1 (DAPI : tem-plate) molar ratio
UV melting experiments Optical melting experiments were performed on a PC controlled PerkinElmer Lambda Bio20 double beam spec-trophotometer equipped with a programmable Peltier temperature-controlled cell holder Quartz cuvettes of
1 cm path length were used and closed with a Teflon cap incorporating the temperature sensor A layer of paraffin was also placed on top of the sample solutions to prevent solvent evaporation Absorbance of each DNA sample was
0.25 per mL, in 10 mMsodium phosphate (pH 7.00) and
100 mMNaCl Complexes were prepared at a base to drug molar ratio of 3.5 Before thermal melting analysis, samples were heated to 80C for 5 min and then slowly cooled to the starting temperature of 3C Melting profiles were acquired by heating samples from 3C to 85 C at a rate of
1CÆmin)1and measuring absorbance corresponding to the maximum around 260 nm at 25C To avoid condensation
of water vapor at low temperatures, the cuvette-holding chamber was dried by flushing with N2gas The melting temperature (Tm) was estimated from the maximum of the first-derivative curve of the absorbance vs temperature Primer extension experiments
Chain extension reactions were catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I (exo-) in
a volume of 20–40 lL containing 8 lM template, 8 lM
primer, 200 lM dNTP, 100 mM NaCl and EcoPol buffer (New England Biolabs) After the addition of primer to the template solutions, samples were denatured at 94C for
4 min and slowly annealed to 37C DAPI was added after primer–template annealing, the solution reactions were incubated with 2.5 U of Klenow fragment at 37C for 1 h and stopped with EDTA to a final concentration of 10 mM Reaction products were desalted by successive dilutions and filtrations using Centricon YM3 (Millipore) (3000 MW
Fig 1 DNA sequences and DAPI chemical structure Schematic
rep-resentation (A) and sequences (B) of the templates and primer used for
chain extension reaction in vitro, catalyzed by Klenow fragment
(3¢ fi 5¢ exo-) of E coli DNA polymerase I The underlined region of
common sequence in (B) represents the primer-complementary region
of the template (C) Chemical structure of DAPI.
Trang 3cut-off) Finally, samples were lyophilized, suspended in
10 lL of formamide and run at 37C and 290 V on a 12%
(w/v) denaturing polyacrylamide gel containing 40% (v/v)
formamide and 7Murea Gels were stained with 0.1% (w/v)
methylene blue in 0.5M sodium acetate Gels containing
5¢-fluorescein labeled DNA were analysed by UV
transillu-mination
Results and discussion
Electrophoretic mobility-shift induced by DAPI
on trinucleotide repeats
Electrophoretic analysis, comparing gel mobility-shift
induced by DAPI on the 20 different individual strands of
trinucleotide repeats, shows an observable increase in
electrophoretic mobility induced by the drug for the triplet
sequence (ATT)6only (Fig 2) As shown previously, such
increased migration is due to the reported ability of the drug
to fold the (ATT)6into a hairpin-like structure by a
minor-groove binding mechanism which stabilizes base pairing of
DNA regions containing TÆT mismatches flanked by AÆT
base-pairs [Ka¼ 3.4 · 106
M )1 considering two binding sites for (ATT)6] [16] The DAPI–(ATT)6 complex is also
the only fluorescent band detected in the electrophoretic gel
It has been reported that binding of DAPI into the
minor-groove of DNA is characterized by a distinctive large
increase of the drug fluorescence quantum yield [25,26] and
by changes in DNA electrophoretic mobility [16,27] As the
electrophoretic bands of (AAT)6, (ACT)6, (AGT)6, (ATG)6
and (ATC)6 do not exhibit such characteristic properties caused by the drug interaction, our results indicate that binding of DAPI to two consecutive AÆT base-pairs flanked
by AÆA, GÆG or CÆC mismatches leads to weaker minor-groove complexes than two consecutive AÆT base-pairs flanked by TÆT mismatches Unexpectedly, even though intercalation mechanisms have been reported for binding of DAPI to GC-rich or mixed sequences [12,13], gel mobility
of the GC-rich triplet is unchanged by DAPI and no fluorescent bands are detected for their DAPI-containing samples, including those triplets that spontaneously migrate faster due to their inherent propensity for self-annealing into hairpin structures: ACG, AGC, CCG, CGG, CGT and CTG (Fig 2) [28] In particular, CGT and CTG triplets, which self-anneal spontaneously by forming two consecu-tive Watson–Crick GÆC base-pairs flanked by TÆT mismat-ches, do not lead to stable complexes with DAPI as in the case of the ATT triplet This indicates the crucial role played
by AÆT base-pairs in addition to TÆT mismatches Therefore, although our electrophoretic results represent the limited case of six triplet sequences, the specificity of the observed effects confirms the higher selectivity of DAPI for the minor-groove of the ATT-triplet with respect to all the other individual strands of trinucleotide repeats Both AÆT and TÆT base-pairs appear to be necessary conditions for determining ATT-regions as the most favourable targets among all the individual strands of trinucleotide repeats
Binding and conformational properties
of DAPI–template complexes The binding effects of DAPI on the replication processes of ATT-triplet sequences were evaluated by comparing prod-ucts of primer extension reactions by three distinct 70-base templates containing, respectively (ATT)10, (AAT)10 or a 30-base long GC-rich random sequence at the 5¢ end, as described in Materials and methods and illustrated in Fig 1A,B The conformational changes induced by DAPI
on the different templates were evaluated to exclude unexpected binding and structural effects due to the presence of common primer-complementary and random sequences As shown in Fig 3, DAPI (50 lM) does not induce appreciable changes in the electrophoretic mobility
of AAT- (lane 6) and GC-rich random-templates (lane 2) Conversely, in the presence of DAPI, the ATT-template migrates faster than its drug-free form (lanes 4 and 3, respectively) This increased gel-mobility induced by DAPI
is not merely attributable to the drug interaction per se, as binding of DAPI normally leads to a reduction in DNA electrophoretic mobility [16,27] Moreover, the relatively low mobility of 1 : 1 AAT-/ATT-template mixture in the presence of DAPI (Fig 3, lane 7) safely excludes the possibility that fast migrating DAPI–ATT-template com-plex could be constituted by dimeric homoducom-plex DNA structures In contrast, the electrophoretic results account for a more compact structure of DAPI-bound ATT-template with respect to its free form, which is consistent with the previously reported lower mobility of the free monomeric random coil structure of (ATT)6in comparison with its hairpin conformer bound to DAPI [16]
Binding mechanisms of DAPI and their effects on the stability of template intramolecular base-pairing were
Fig 2 Effect of DAPI on the electrophoretic mobility of trinucleotide
repeat DNA sequences (A) Nondenaturing 12% polyacrylamide gel
migration of the 20 possible triplet repeat strands and their complexes
with DAPI at 4 : 1 drug/template molar ratio (B) Before gel
inocu-lation, samples were denatured at 90 C and slowly annealed to room
temperature Drug–DNA complexes were prepared by adding DAPI
following DNA annealing Gels were stained with 0.1% (v/v)
methy-lene blue in 0.5 M sodium acetate Samples were 35 l M DNA and
150 l DAPI in Tris/borate/EDTA buffer.
Trang 4evaluated by fluorescence and UV thermal melting studies.
The addition of ATT-template to DAPI solution determines
drug fluorescence changes that are distinctive of the drug
binding into the minor-groove of the double-helix structure
of AT-rich sequences [25]: the drug fluorescence signal is
strongly increased (8.3-fold at a drug/template molar ratio
of 2 : 1) and the maximum emission is shifted to lower
wavelengths (20 nm) (Fig 4) The observation of such
distinctive fluorescence properties of the minor-groove
binding mechanism implies the presence in the DAPI–
ATT-template complex of a double-helix structure, in
agreement with the hairpin suggested by the increased gel
mobility of the DAPI–ATT-template complex In contrast,
addition of AAT- and GC-rich random-template oligomers
to the drug solution causes changes in DAPI fluorescent
properties which are indicative of a weak minor-groove
interaction, GÆC-like intercalation or uncharacterized
bind-ing mechanisms: the shift in the maximum emission at a
2 : 1 drug/template molar ratio was about 12 and 15 nm,
and the increase in the fluorescence intensity was only
2.2- and 1.6-fold for AAT- and random-template,
respect-ively (Fig 4)
UV thermal melting studies are also consistent with a
double-helix structure of ATT-template complexed with the
drug Binding of DAPI strongly increases the melting
temperature of the ATT-template from 17C to 45 C as
measured by the shift in sigmoidal melting profiles
illustra-ted in Fig 5 Consequently, at the Klenow reaction
temperature of 37C, the drug-free ATT-template is almost completely in random coil conformation (Tm¼ 17 C) whereas its DAPI-bound form is prevalently base-paired (Tm¼ 45 C) Moreover, along with a single electropho-retic band produced by the ATT-template in the presence of DAPI (Fig 3, lane 4), the observation of a monophasic thermal transition is consistent with the prevalent presence
of a single DNA structure in the complex, which excludes the observable presence of dimeric homoduplex structures
Fig 3 Effect of DAPI on the electrophoretic mobility of DNA
tem-plates Nondenaturing 12% (w/v) polyacrylamide gel migration of
GC-rich random-template (lanes 1 and 2), ATT-template (lanes 3 and
4), template (lanes 5 and 6) and 1 : 1 mixture of ATT- and
AAT-template Samples with (+) and without (–) DAPI are indicated.
Before gel inoculation, samples were denatured at 90 C and slowly
annealed to room temperature Drug–DNA complexes were prepared
by adding DAPI following DNA annealing Gels were stained with
0.1% (w/v) methylene blue in 0.5 M sodium acetate Samples were
8 l M template–primer and 50 l M DAPI in Tris/borate/EDTA buffer.
Fig 4 Effect of DNA templates on the fluorescence spectrum of DAPI DAPI fluorescence spectra in (a) the free form, in the presence of (b) random-template, (c) AAT-template and (d) ATT-template at 2 : 1 drug/template molar ratio The experiments were performed at 37 C,
in 100 m M NaCl and 10 m M phosphate buffer at pH 7.00 Excitation (350 nm) and emission (448 nm) with 1.5 nm band-pass were used and spectra were corrected for background signal.
Fig 5 Effect of DAPI on the melting curve of ATT-template Melting curves of ATT-template in free (- - -) and DAPI-bound forms (––) at a base/drug molar ratio of 3.5 The DNA solution absorbance was 0.25 per mL in 10 m M sodium phosphate (pH 7.00) and 100 m M NaCl, and quartz cuvettes of 1 cm path length were used Samples were heated at
a rate of 1 CÆmin)1and absorbance was measured corresponding to the maximum around 260 nm, at 25 C Hyperchromicity (%) at the temperature T C was given by [A (T C) – A (3 C) ]/A (3 C) , where A (T C)
and A (3 C) represent the A 260 at T C and 3 C, respectively.
Trang 5in addition to the monomeric hairpin In conclusion, the
spectroscopic and electrophoretic results shows that the
common random GC-rich sequences of the ATT-template
as well as the increased length of its triplet sequence, do not
prevent its (ATT)10 region from adopting a hairpin-like
structure the equivalent of that induced by DAPI in the
(ATT)6oligomer [16]
Primer extension experiments
DNA polymerization studies for evaluating the biological
effects of the binding of DAPI to ATT-, AAT- and
random-template sequences were performed by the Klenow
frag-ment of DNA polymerase I using a 15-base primer
complementary to the 41–55 base-pair region of the
70-base templates reported in Fig 1 The fully extended
product of Klenow synthesis was 15 bases shorter than the
templates due to the 15 unpaired bases at the 3¢-end of the
templates annealed to the primer (Fig 1) As shown by
electrophoresis (Fig 6), at the 16 : 1 DAPI/template molar
ratio, the expected fully extended 55-base product of
Klenow synthesis is present in the lane containing the
reaction products of AAT-template In contrast at the 16: 1
DAPI/template molar ratio, DAPI inhibits synthesis of the
55-base product in the ATT-template directed reaction
Such a sequence-dependent inhibition was effective when
the DAPI/template molar ratio is equal to 8 (DAPI/
(ATT)2¼ 1.6), while full-length synthesis products are
observed at a drug/template molar ratio of 4 (DAPI/
(ATT)2¼ 0.8), although DAPI/template values of both
4 and 8 present visible products of partial synthesis
The experiments in Fig 6were performed at a constant
template concentration of 8 lM and drug concentration
ranging from 8 to 128 lM Such a tight binding condition
(Ka¼ 3.4 · 106
M )1 considering two binding sites for
(ATT)6[16]) strongly shifts the binding equilibrium towards
the bound species This indicates that DAPI strongly
increases its inhibitory activity when complexes are formed
at a stoichiometric molar ratio of around 1 : 1 with (ATT)2
target sites
The observed dependence on template sequence excludes the possibility that the inhibitory property of DAPI could only be attributed to a direct interaction of the drug with the Klenow fragment A DNA-mediated step correlating with the different structural and binding characteristics of DAPI–template complexes is involved The lanes of Fig 6 relating to synthesis products using the AAT-template (8 lM), show a weak inhibitory activity of DAPI at high concentration (128 lM), which is more evident at the highest drug concentration (256 lM) (Fig 7) Such a weak inhib-itory activity on the AAT-triplet is also sequence-dependent
At the highest concentration (256 lM), DAPI has a very weak effect on the random-template synthesis appearing to
be almost ineffective on GC-rich sequences (Fig 7) This finding is also supported by the observation that the first 10 bases of the GC-rich random sequence of the AAT- and ATT-templates are copied without any pausing in relation
to the drug-free syntheses This provides evidence for a relatively weak sequence-independent inhibitory effect of DAPI on Klenow activity at our experimental conditions Therefore, inhibitory activity of DAPI appears to be significantly effective only for the AT-rich sequences of the AAT- and ATT-templates, even though with different characteristics, as it appears weaker and more homogeneous along AAT- than ATT-triplet sequences The weak inhibi-tion at AAT-triplet sites is attributable to a binding mechanism that presents spectroscopic characteristics that are different from those reported for intercalation and minor-groove binding, indicating minor involvement of DNA bases [16] However, the absence of a comparable inhibitory effect at GC-rich random sequences indicates a significant direct or indirect involvement of AAT bases in
Fig 6 Effects of different DAPI/template molar ratios on Klenow
synthesis Denaturing gel analysis showing the effect of DAPI on
Klenow-catalyzed 5¢-fluorescein labeled primer elongation by
ATT-and AAT-template Reactions were performed at the constant
tem-plate concentration of 8 l M with different DAPI/template molar ratios
(R) Ladder is constituted by 5¢-fluorescein-labeled DNA oligomers
with sequences similar to those expected for the reaction products of
the ATT-template.
Fig 7 Effect of high DAPI concentration on template-directed Klenow synthesis Denaturing gel analysis showing the effect of high DAPI concentration on the elongation of 5¢-fluorescein labeled primer by ATT-, AAT- and random-template-directed Klenow synthesis Sam-ples were 8 l M primer–template and 256 l M DAPI Presence (+) and absence (–) of DAPI in the reaction solution is indicated Ladder is constituted by 5¢-fluorescein labeled DNA oligomers with sequences similar to those expected for the reaction products of the ATT-template.
Trang 6the interaction with DAPI This suggests an efficient
mechanism of single-stranded sequence recognition
charac-terized by small changes in NMR resonances of DNA bases
[16] It is also relevant that the intercalation mechanism
reported for the binding of DAPI at AÆU [25] and GÆC [12]
sites does not appear to significantly affect Klenow activity
catalyzed on the AAT- and GC-rich random-templates The
synthesis was stalled by DAPI on the ATT-bearing template
giving rise to a prevalent product of about 31–33 bases
(Fig 7) indicating that, in addition to the initial 10-base
random sequence, at least 6–8 bases of ATT-region are
copied by the Klenow fragment before it stalls In other
words, the ATT motif itself does not appear to be sufficient
to block DNA synthesis by DAPI, suggesting an effect by
different structural features along the (ATT)10 sequence
caused by the drug interaction As shown above, DAPI
induces the formation of hairpin structures on the ATT- but
not on the AAT- and GC-rich random-templates In
addition, it has been reported previously that long tracts
of CTGÆCAG repeats, which have the propensity to fold
into stable hairpin structures [29–31], block the replication
fork in E coli [32] and cause a pause in DNA synthesis
in vitro[33] These findings suggest that the stall of Klenow
progression observed in the present study could be
attrib-uted to the (ATT)10 region (located at the 5¢-end of the
ATT-template) folding into a hairpin-like structure The
double helix of the hairpin stem, stabilized by the binding of
DAPI into the minor-groove of AÆT and TÆT base-pairs,
appears to be responsible for stalling the Klenow fragment
As AÆA and TÆT mismatches cause similar destabilizing
effects on the double helix structure [34–36], the dissimilar
influence of DAPI on the Klenow catalyzed replication of
AAT- and ATT-containing sequences can only be
attrib-uted to the more favourable characteristic of the TÆT
minor-groove than the AÆA minor-minor-groove, in the binding of DAPI
[16] In conclusion, the main cause of the strong inhibitory
effect of DAPI on DNA replication in vitro reported in this
work appears to be linked to the propensity of the drug to
stabilize the double helix structure by favourable
electro-static, H-bonds and van der Waals contacts with the DNA
minor-groove surface [11,16] Although it should be
men-tioned that the drug could affect other cellular processes at
the conditions necessary for stalling DNA polymerase
progression, the results nevertheless suggest a novel
struc-tural model for the molecular mechanism of the action of
minor-groove binding ligands
Conclusion
This study reports the effect of the classical minor-groove
binding ligand DAPI on AATÆATT trinucleotide repeat
replication in vitro, catalyzed by the Klenow fragment of
DNA polymerase I The high affinity and structural
changes selectively produced by DAPI on the ATT-strand
of the AATÆATT trinucleotide repeat are associated with
the stalling of Klenow progression along the ATT-template
sequence This draws attention to the biological relevance
of high affinity drugs with structural effects on
single-stranded DNA and RNA The main genetic processes such
as replication, transcription and translation involve
indi-vidual strands of DNA and RNA, emphasizing the
importance of studies that attempt to clarify the
relation-ship among binding, structural and biological aspects relating to this conformational class of nucleic acids Particularly in the case of trinucleotide repeats, the secondary structures of individual strands are considered
to be the main cause of their expansion, and are associated with a number of human genetic diseases [28] In particular, the formation of stable intramolecular hairpins appears to be the most probable cause of CAGÆCTG and CGGÆCCG expansion, by favouring slippage of DNA strands during DNA replication We have found previ-ously that the spontaneous hairpins reported for the T-rich strand of CAGÆCTG triplets are similar to those induced
by DAPI and Hoechst on the T-rich strand of AATÆATT trinucleotide repeats [16] The present work shows that such a structural affinity is associated with a comparable ability to influence DNA polymerase activity, given that tracts of CAGÆCTG triplets can affect DNA replication
in vivo[32] and can be sites of pausing of DNA synthesis
in vitro [33] The CAGÆCTG triplet repeats present additional biological characteristics that have been attrib-uted to its propensity to fold into hairpin structures, suggesting further potential biological properties of DAPI-induced hairpins: regions of CAGÆCTG triplets are tran-scribed significantly slower than random sequences [37] and can elude cellular mechanisms designed to repair DNA [38] Moreover, because it has emerged that specific DNA sequences within introns can regulate transcription [19,20], the prevalent location of ATT triplet repeats within introns in the human genome [18] suggests strongly that binding of DAPI to ATT-bearing genomic targets may influence the transcriptional processes
In conclusion, the results reported in this work suggest a novel molecular mechanism of action of the classical minor-groove binding ligand DAPI This property is associated with the ability of the drug to bind and stabilize folded structures in ATT-triplet sequences, by favourable and selective interaction with the minor-groove of AÆT and TÆT base-pairs The molecular mechanism proposed in this work may help in designing new minor-groove binding ligands with sequence-selective activity on DNA replication
Acknowledgements
We thank Fabio Bertocchi for his technical assistance This work is supported in part by target project Biotecnologia of Italian CNR.
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