In the present work, we demonstrated that NAPs protect naked genomic DNA from DNase I, whereas natural polyamines spermine, sper-midine and putrescine fail to do so.. We believe that NAP
Trang 1structures that play a crucial role in genomic DNA
protection and conformation
Luciano D’Agostino1, Massimiliano di Pietro1and Aldo Di Luccia2
1 Department of Clinical and Experimental Medicine, ‘Federico II’ University of Naples, Italy
2 Department of Animal Production, University of Bari, Italy
Polyamines interact with DNA phosphate groups by
means of nonspecific electrostatic bonds [1] This
inter-action has been shown to result in the protection of
small DNA molecules from common damaging agents,
such as ionizing radiation and reactive oxygen species
[2,3] Polyamines in solution with polynucleotides have
also been shown to inhibit the activity of
endonucle-ases, including DNase I [4–7] The protective ability of
polyamines is attributable not only to the formation of
a steric barrier against DNA-damaging agents, but
also to their property to condense the DNA In fact,
polyamines, like other cations, induce DNA
condensa-tion as a consequence of the inhibicondensa-tion of > 90% of
DNA negative charges [8] Analogous in vivo
experi-ments have demonstrated that spermine and, to a
lesser extent, spermidine, prevent DNA
fragmenta-tion and the onset of apoptosis Protecfragmenta-tion from
enzymatic cleavage appears to be the result of a
modi-fied chromatin arrangement, rather than inhibition of
the endonuclease activity [9]
Condensation of DNA in the presence of poly-amines has also been proposed to be instrumental in genome packaging [10] This should be regarded of crucial importance if we consider that the total length
of cellular DNA is 1 m, whereas the size of the nuc-leus is in the range of several micrometers [11] How-ever, condensation should not be considered as a static state, as the elasticity is a mechanical property of the DNA, indispensable to cellular processes such as repli-cation and transcription [12,13] For these reasons the DNA strands in vivo, at the same time, must be pack-aged and protected, but not restrained
The structural impact of polyamines on DNA is also supported by the evidence that these compounds induce, on polynucleotides, a transition from the right-oriented B-form to the left-handed Z-form [14,15] Such an effect might be important for DNA physiol-ogy, as a tight connection occurs between transcrip-tional activity on DNA and the acquisition of a Z-form [16]
Keywords
DNA conformation; DNA protection;
molecular aggregates; polyamines;
supra-molecular chemistry
Correspondence
L D’Agostino, MD, Facolta` di Medicina
‘Federico II’, Edificio-6, Via S Pansini, 5,
80131 Naples, Italy
Fax: +39 081746 2707
Tel: +39 081746 2707
E-mail: luciano@unina.it
(Received 4 April 2005, accepted 19 May
2005)
doi:10.1111/j.1742-4658.2005.04782.x
In a previous study we showed that natural polyamines interact in the nuclear environment with phosphate groups to form molecular aggregates [nuclear aggregates of polyamines (NAPs)] with estimated molecular mass values of 8000, 4800 and 1000 Da NAPs were found to interact with genomic DNA, influence its conformation and interfere with the action of nucleases In the present work, we demonstrated that NAPs protect naked genomic DNA from DNase I, whereas natural polyamines (spermine, sper-midine and putrescine) fail to do so In the context of DNA protection, NAPs induced noticeable changes in DNA conformation, which were revealed by temperature-dependent modifications of DNA electrophoretic properties In addition, we presented, for NAPs, a structural model of polyamine aggregation into macropolycyclic compounds We believe that NAPs are the sole biological forms by which polyamines efficiently protect genomic DNA against DNase I, while maintaining its dynamic structure
Abbreviation
NAP, nuclear aggregate of polyamines.
Trang 2A body of evidence also indicates a role for
poly-amines in the regulation of gene expression [17] and in
the cell cycle A correlation has been shown between
increased concentrations of spermine and spemidine in
the nucleus and the induction of mitosis [18–20]
Moreover, alterations in the polyamine biosynthetic
pathway affect the correct progression of the cell cycle,
particularly the S-phase [21]
Temperature is an additional factor capable of
affecting DNA conformation It has been reported
that (a) an increase of a few C is associated with a
reduction in the overstretching forces of the DNA
strands [22,23], (b) that the DNA melting
tempera-ture is directly correlated with the chain length of
interacting polyamines [24–27] and (c) that a
trans-ition of the DNA chain from a dispersed coil state
to a condensed-collapsed state parallels an increase
in temperature [28] These findings draw attention to
the relationship existing between temperature and
stabilization, aggregation, elasticity and
conforma-tional transition of the DNA, all phenomena closely
linked to DNA protection and influenced by
poly-amines
Recently we described new compounds with
mole-cular mass values of 1000, 4800 and 8000 Da, named
nuclear aggregates of polyamines (NAPs), whose
molecular structure is based on the ionic interaction
between polyamines and phosphate groups These
compounds were isolated from the nuclei of several cell
types In vitro aggregation experiments demonstrated
that by mixing polyamines (spermine, spermidine and
putrescine) in phosphate buffer it is possible to
gener-ate compounds with molecular mass identical to the
NAPs extracted from cells [29] This finding suggested
that NAPs can form naturally in the nuclear
environ-ment, where phosphates are particularly abundant
The positive net charge of NAPs allows interaction
with the negatively charged DNA phosphates We
have shown previously that these compounds influence
DNA conformation and protect DNA from
Exonuc-lease III and DNase I [29]
The compound with a molecular mass of 4800 Da,
which was suggested to induce supercoiled DNA
forms, is clearly associated with cell replication, being
recovered in large quantities in the nuclei of cells
in S-phase and absent in non-replicating cells
Experi-mental evidence indicates the NAP with the lowest
molecular mass (1000 Da) functions as a precursor for
the 4800 Da form of NAP The concentration of the
8000 Da form of NAP in the nucleus did not vary
throughout the phases of the cell cycle
In this study, based on the investigation (by gel
elec-trophoresis) of the interaction between polyamines and
genomic naked DNA, we compared the protective efficacy of NAPs with that of single polyamines against DNase I We demonstrated that only NAPs efficiently impede nuclease cleavage, suggesting that they are the biologically effective forms by which poly-amines protect the genomic DNA from endonucleases
We propose a model of molecular organization of polyamines into NAPs extrapolated from our previous and present experimental evidence
NAPs were previously named according to their molecular mass However, as the molecular mass was only an approximate value, estimated by GPC analysis and therefore different from that calculated by the molecular formulas presented here, we now adopt, in the present study, an alternative nomenclature based
on size Therefore, < 1000 NAP, 4800 NAP and 8000 NAP will subsequently be named s-NAP, m-NAP and l-NAP (small-, medium- and large-sized), respectively
Results and Discussion
Single polyamines fail to protect naked genomic DNA from DNase I
To assess the protective role of NAPs against endonuc-leases and compare it with that of single polyamines,
we carried out electrophoretic assays of genomic DNA treated with DNase I Single polyamines or NAPs were allowed to interact with genomic DNA before expo-sure to phosphodiesterasic endonuclease Assuming that the tested compounds, interacting with DNA, pre-vent DNA degradation, the protective effect can be demonstrated by a higher molecular mass of the DNA molecules migrating into the gel
We first tested DNA degradation by DNase I upon incubation with increasing concentrations of single polyamines, starting from a concentration of 1 lm polyamines, which is comparable to the concentration
of polyamines forming NAPs in the extractive elution
As shown in Fig 1A, a 1 lm concentration of poly-amines did not protect from DNase I, and no noticeable increase in DNA protection was observed with 50 or
150 lm single polyamine concentrations Increasing the concentration of single polyamines up to 600 lm resul-ted in no clear impediment to DNA cleavage (Fig 1B)
At 600 lm spermine, a peculiar effect was observed, namely the paradoxical facilitation of DNase I, result-ing in the complete degradation of DNA (Fig 1B, lane c) This phenomenon, which probably depends on the electrostatic nature of the polyamines–DNA inter-action, is in accordance with previously published results indicating a biphasic behavior of DNA structure
in water solution with increasing concentrations of
Trang 3spermine [30] The authors of this study observed that
spermine induces DNA condensation and precipitation
as a consequence of the progressive neutralization of
the negative charges of DNA This phenomenon was
attributed to the organization of DNA into a
liquid-crystalline ordered structure determined by the
interac-tion of a single polyamine with several negative DNA
charges However, DNA resolubilization occurred
when spermine was supplemented in excess DNA
res-olubilization was attributed to the osmotic stress
gen-erated by the high concentration of polyamines, which
drives these cations into the nonpolar DNA phase,
and to the decreased number of DNA-binding sites
per polyamine, making the DNA more hydrophilic
Analogously, in our experimental model, high levels of spermine in solution rendered the genomic DNA extre-mely sensitive to the action of DNase I We believe that this is a result of the resolubilization of DNA and the massive exposure of the phosphodiester bonds to the active site of the nuclease, probably because of repulsive effects exerted by polyamines present in the nonpolar phase of the DNA on polyamines linked to the backbone phosphates
As single polyamines did protect naked genomic DNA from DNase I, we investigated whether natural aggregates of polyamine, such as NAPs, might possess this ability As shown in Fig 2 (left panel), all NAPs protected DNA from degradation, although the migra-tion pattern of DNA preincubated with l-NAP differed from that of DNA preincubated with either m-NAP or s-NAP
As polyamines were not only ineffective in DNA protection, but even detrimental for DNA integrity
at higher concentrations, aggregation into NAPs may reflect the need to keep the concentration of intra-nuclear polyamines at low levels and under stringent control In fact, other studies have demonstrated that
an excess of polyamines may result in the perturbation
of vital functions dependent on DNA integrity and conformation [31], whereas their drastic decrease under the lower threshold can impede cell mitosis and⁄ or trigger the mitochondria-mediated apoptotic pathway [32] In order to accomplish this tight regulation that hampers an excessive increase of polyamines in the
A
B
Fig 1 Electrophoresis of genomic DNA preincubated with single
polyamines and then exposed to DNase I (A) Electrophoretic
migration, at 37 C, of genomic DNA preincubated with three
differ-ent concdiffer-entrations (1, 50 and 150 l M ) of spermine (lanes c, c¢ and
c¢¢), spermidine (lanes d, d¢ and d¢¢) or putrescine (lanes e, e¢ and
e¢¢) and then exposed to DNase I Whole genomic DNA (lane a)
and DNase I-digested genomic DNA (lane b) were controls (B)
Electrophoretic migration, at 37 C, of genomic DNA preincubated
with 600 l M spermine (lane c), spermidine (lane d) or putrescine
(lane e) and exposed to DNase I Controls were in lanes a (whole
genomic DNA) and b (DNA exposed to DNase I) Identical results
were obtained at a migration temperature of 40 C (data not
shown).
Fig 2 Nuclear aggregates of polyamines (NAPs) protect genomic DNA from DNase I and, at the same time, influence DNA conforma-tion The electrophoretic migration at 37 C (left) and 40 C (right)
of genomic DNA preincubated at 37 C with small-size NAP (s-NAP; lanes c and c¢), medium-size NAP (m-NAP; lanes d and d¢) or large-size NAP (l-NAP; lanes e and e¢) and exposed to DNase I Controls were the whole genomic DNA (lane a) and the DNA fully digested
by DNase I (lane b) The DNA 1 kb ladder marker was in lane m.
Trang 4nucleus, cells are also provided with enzymes that
interconvert and catabolize polyamines [33,34] A key
enzyme for polyamine catabolism is diamine oxidase,
which is able to bind the DNA and oxidize
DNA-bound polyamines [35] The fact that this enzyme is
particularly evident in differentiated enterocytes [36–
38], which are involved in the uptake and distribution
of polyamines [39] to the entire organism via the
intes-tinal bloodstream, suggests that it probably has the
strategic function of coping with the flow of
poly-amines potentially harmful to the DNA of intestinal
cells We believe that NAPs are an important part of
this physiological scenario
NAPs protect DNA in the context of structural
elasticity
The data shown in Fig 2 (left panel) indicated not
only that NAPs preserve naked genomic DNA from
DNase I-dependent degradation with an efficacy much
greater than that of single polyamines, but also that
the migration patterns of NAP–DNA complexes differ
substantially Namely, the DNA preincubated with
l-NAP showed a diffuse migration pattern, whereas the
DNA interacting with s-NAP and m-NAP migrated in
a compact form similar to that of naked DNA (lane
a), but significantly faster Hence, we wondered
whe-ther such a change in DNA migration properties was
caused by a difference in the protection ability of
sin-gle NAPs or by conformational changes induced in
DNA by the interaction with NAPs As temperature
has been shown to be a variable that dynamically
influences DNA conformation in terms of elasticity
and condensation status [28], we varied, within
physio-logical ranges, the temperature of the electrophoretic
run We believed that this modification of the
experi-mental conditions, taking place after inactivation of
the endonuclease, could influence migration changes
based on DNA conformation, but not on the
differen-tial degradation of nucleic acids By raising the
run-ning temperature from 37C to 40 C, we observed a
mirroring change of the DNA electrophoretic patterns
(Fig 2, right panel)
Many aspects of the experiments described in Fig 2
are worthy of discussion First, upon incubation with
each NAP, DNA, although exposed to the
endonuc-lease, showed migration features that, at least in one
of the thermal conditions applied, were not dissimilar
to that of control DNA This result undoubtedly
dem-onstrates that all NAPs, independently of their
molecular mass and net charge, completely protect
DNA from DNase I We believe that the protection
occurs as a result of steric hindrance of DNase I access
to the DNA phosphodiester bond even though we cannot exclude that modification of the DNA conden-sation status might play an additional role Theoretic-ally, the possibility exists that the protection of DNA
by NAPs depends on modification of the catalytic properties of DNase I, rather than by preventing access of DNase I to the DNA phosphodiester bonds However, the latter seems to be the likeliest possibility,
as the protection of DNA from nucleases is a general property of NAPs, regardless of the type of nuclease tested (NAPs have been shown to prevent exonuclease III – another type of nuclease – degradation of DNA [29]) Furthermore, it has already been suggested that spermine prevents in vivo endonuclease activity as con-sequence of a modified degree of chromatin accessibil-ity to the enzyme [9]
Second, all NAPs increased the electrophoretic speed
of genomic DNA, up to induce a diffuse migration pattern, which was determined by each NAP selec-tively at a given temperature (either 37 or 40C) As temperature modifications always followed DNase I inactivation, we were able to exclude any interference
of this environmental change with the enzymatic activ-ity Therefore, we concluded that, in the context of constant DNA protection, NAPs interfere with the DNA condensation status in a temperature-dependent manner We believe that enhancement of the migration speed is attributable to DNA decondensation and strand elongation, which facilitates the penetration of DNA into the gel matrix [40,41]
A third aspect concerns the relationship between DNase I and DNA conformation Our experiments suggest that the incubation of NAPs–DNA complexes with DNase I was a decisive factor in the above men-tioned conformational effects, as NAPs alone deter-mined only slight modifications in the running properties of DNA [29] It has already been shown that single polyamines, mainly spermine, can modulate the DNA-binding properties of proteins, either increas-ing or diminishincreas-ing their affinity for DNA [42] This ability, to modulate the DNA-binding properties of proteins, was analyzed in relation to the conformational effects of polyamines on DNA and was found to be directly dependent on the degree of their positive charge Accordingly, we hypothesize that, in the pres-ence of NAPs, DNase I, while prevented from acting
as a nuclease as a result of steric inhibition, interacts with NAPs–DNA complexes and, in turn, cooperates with NAPs to modify DNA arrangement Ionic forces may drive the interaction between DNase I and NAPs–DNA complexes In fact, under the experi-mental conditions applied (inactivation by EDTA and the electrophoretic run performed at pH 8), the
Trang 5enzyme acquires a negative net charge and can
there-fore bind the positively charged NAPs
In contrast to polyamines, the effects of NAPs on
DNA condensation status do not seem to depend
mainly on DNA charge neutralization By virtue of
their net charge, single polyamines have been shown to
influence the conformation of high Mr DNA [28,41]
Namely, T4 DNA in buffer solution acquired an
elon-gated coiled conformation, whereas the progressive
neutralization of the global DNA charge by interacting
polyamines determined the acquisition of a
compact-folded conformation, which showed a slower
electro-phoretic mobility [41] Differently, the modification of
the electrostatic properties of DNA induced by NAPs
does not appear to be a primary factor driving the
change of condensation status, as the different
migra-tion patterns were obtained without altering the
con-centration of NAPs in the solution The marked DNA
electrophoretic changes indicate, in our opinion, that
NAPs efficiently preserve DNA elasticity and can
modulate the degree of DNA strand elongation, which
is measured by mobility on the gel These findings
sug-gest a possible role for NAPs in the in vivo nuclear
environment: NAPs-dependent modification of the
DNA condensation status might play a role in the
regulation of chromatin complexation onto histones
The elasticity of the DNA was correlated to both
its interaction with polyamines [43] and temperature
Melting experiments demonstrated that polyamines
stabilize the DNA structure with an ability that is a
function of the polyamine chain length [24–26]
More-over, the melting entropy of DNA was determined by
measuring the overstretching force of single molecules
of DNA [22,23] This transitional force decreases with
the increase of temperature from 11 to 52C, thus
indicating that the stability of the DNA double helix is
a temperature-dependent phenomenon and that DNA
melting occurs during the overstretching transition
However, the maintenance of an appropriate
func-tional morphology of the DNA seems to require more
complex mechanisms Studies of cation interactions
indicate that the size of the DNA grooves depends on
the number of charges present on the DNA backbone
In fact, the repulsion of phosphate groups across the
minor groove makes it widen, whereas the
neutraliza-tion of the phosphate groups reduces the groove width
[44,45] Even though the groove’s flexibility is crucial,
its collapse [46] should be considered a detrimental
event that can be more efficiently prevented by the
interaction with the l-NAPs rather than with the small
sized polyamines Our conviction is strengthened by
the fact that the collapse of high Mr DNA to toroidal
and spheroidal structures has been reported in the
presence of multivalent cations, including spermidine and spermine [46] Recent DNA thermodynamic stud-ies also support this belief, as they indicate that the distension of strands caused by temperature increases widen the grooves [22,23], so permitting the interaction
of larger moieties
Therefore, two implications can be inferred from our results (a) the preservation of the DNA integrity is fully assured by NAPs along with the modification of the folding state of the DNA and (b) a few degrees of temperature increase, a normal occurrence in living cells, is able to drive significant conformational chan-ges in the presence of NAPs and DNase I Both of these events imply that NAPs carry out their defensive function against DNase I having constantly full acces-sibility into the DNA grooves
For all of these reasons, NAPs, compounds with an optimal mass⁄ charge ratio, represent supramolecular structures able to determine a broader impact on DNA structure and physiology than single polyamines
A model of polyamine aggregation into NAPs
As a last step of the present study, we sought to pro-pose a structural model of polyamine organization into NAPs in accordance with the experimental data pro-duced to date about NAPs biochemistry (summarized
in Table 1), and the theoretical principles of macro-molecule self-assembly (see the Experimental proce-dures)
The NAPs were drawn as macro(poly)cyclic com-pounds on the basis of the following assumptions (a) the attraction of opposite charges of polyamines and phosphate represents the driving force of self-assembly, (b) the intercalation of a phosphate anion between the N-terminal ends of two polyamines per-mits the formation of a cyclical structure character-ized by a minimal repulsive force as a consequence
of thermodynamic and kinetic stability and selectiv-ity, (c) ion water solvatation takes part in the supra-molecular aggregation, conferring high flexibility to ionic bonds, (d) amine nitrogen of polyamines, com-pletely protonated at physiological pH, cannot parti-cipate in the formation of hydrogen bonds, whereas phosphate groups are able to form hydrogen bonds, and (e) hydrogen bonds among phosphate groups stabilize adjacent polycyclic units into tridimensional supramolecular structures
The binding of phosphate groups to polyamines to form cyclic supramolecules can overcome the mere phenomenon of attraction of charges to imply the pro-cess of molecular recognition, already described more than 20 years ago [47] Our previous NMR studies
Trang 6showed that phosphate groups which interact with
long polyamines (spermidine and spermine), are able
to determine molecular rearrangements in their
struc-ture [29] These modifications might include expression
of enhanced flexibility on the major axis of
poly-amines, which favours their bending, and would be
instrumental to the formation of cyclic supra-molecules
A cyclical structure of NAPs has already been
sugges-ted in view of their absorbance peak at 280 nm [29],
which is compatible with an electron delocalization
typical of molecules with pfi p bonds, such as
poly-ene systems Moreover, it has already been postulated
that macropoycyclic compounds possess a structure
favourable for maximizing and optimizing functional
molecular activities Such molecules are usually large
(macro), and may therefore contain central cavities and
possess numerous bridges and connections (polycyclic)
[47] Importantly, the formation of polyamine-based
macrocyclic compounds has already been described,
either as a spontaneous biological event [48], or as a
result of in vitro synthetic experiments [49]
The s-NAP, whose molecular mass, extrapolated
from the simplest formula, is 1000 Da, is composed
of two spermines, one spermidine and one putrescine
As a result, we can predict its structure to be a single
tetrameric ring formed by four polyamines linked by
four phosphate groups through ionic bonds (Fig 3A)
We have previously shown that, in synchronized
Caco-2 cells stimulated to replicate by gastrin, the
diminu-tion of the s-NAP pool was accompanied by
increased levels of m-NAP This observation raised the
hypothesis that s-NAPs have the property to aggregate
into larger molecules (i.e m-NAPs) [29] Further
sup-port of this hypothesis came from the detection of
compounds with intermediate molecular mass values
(ranging from 1000 to 4800 Da) in the above
mentioned in vitro aggregation studies [29] Further-more, previous biochemical studies indicated that m-NAP conserves the same spermine⁄ spermidine ⁄ putrescine ratio (2 : 1 : 1) of s-NAP For all of these reasons, and because the molecular mass of m-NAP was estimated to be 4800 Da [29], we proposed its structure to consist of five 4-polyamine monomers con-nected by hydrogen bonds (Fig 3B)
The largest NAP (l-NAP) has a polyamine ratio of
1 : 1 : 1 [29] In contrast to m-NAP, we did not isolate
a pool of monomers for the formation of l-NAP, therefore its structural model results are more specula-tive However, following the criterion of analogy with the smaller compounds (s-NAP and m-NAP), we pre-dict that its structure may originate from the aggrega-tion, by hydrogen bonds, of five 6-polyamine rings (i.e two spermines, two spermidines and two putrescines) linked by phosphate groups (Fig 3C) We predicted 6-polyamine rings in the structure as (a) 3-polyamine rings are likely to require an excessive degree of poly-amine bending and a high energetic level, which would give rise to a less favourable structure, and (b) larger rings (of 9-polyamine units or larger) would be extre-mely large for fitting into the DNA grooves where NAPs are believed to interact
The primum movens in NAPs–DNA aggregation is the charge attraction between DNA phosphates and the amino groups of polyamines As the amino groups
of polyamines are already engaged in ionic bonds with the phosphates of NAPs, secondary amino groups are those available to establish interstrand interaction with the backbone phosphates In accordance with a recently proposed model of spermine interstrand com-plexation along the major groove, we believe that the interaction of NAPs rings with DNA is then stabilized
by intra major groove bonds with DNA bases They
Table 1 Experimental data supporting nuclear aggregates of polyamines (NAPs) modelling Put, putrescine; Spd, spermidine; Spm, spermine;
Ph, phosphate group.
a Gel permeation chromatographic analysis of 25 l M polyamines (Spm, Spd and Put) dissolved in phosphate buffer (pH 7.2) b UV spectro-photometric detection at 280 nm c Defined on the basis of the molar concentration of polyamines forming NAPs d Gel permeation chroma-tographic analysis. eOn formulae shown in Fig 3. fDiameters can vary owing to the flexibility of the electrostatic interactions linking polyamines and phosphate groups g Speculative hypotheses.
Trang 7can be either of the hydrophobic type (between the
CH2 group of thymine and the methylene groups of
spermine) and⁄ or the ion-dipole type (between the
sec-ondary amino groups of polyamines and purine-N7
or thymine-O4 residues) [1,50] The insertion of NAP
monomers into the DNA grooves forms the basis of
the recognition process occurring between the two
supramolecular structures (i.e DNA and NAPs) In
our model, the adaptation of both l-NAP and m-NAP
to the DNA shape was hypothesized to be favoured by
bidirectional movements of the arms of an arch-like
structure (Fig 3B,C) Such an event is made possible
by the hydrogen bonds between phosphates belonging
to contiguous rings that confer great flexibility to the
macropolycyclic structures of NAPs It could be
argued that NAPs, whose structural integrity relies on
weak interactions (electrostatic and hydrogen bonds),
might disaggregate once in contact with DNA
How-ever the results of electrophoretic experiments allowed
us to exclude such a possibility In fact, the loss of
NAPs’ integrity would leave, in solution, single
poly-amines (spermine, spermidine, and polypoly-amines), which
we showed do not possess any relevant DNA
protect-ive activity Therefore, in order to protect DNA from
DNase I, NAPs must maintain their structural
integ-rity Moreover, their ability to influence DNA
confor-mation is a further indirect sign that NAPs must be
not disaggregated when in contact with DNA
It is also conceivable that NAPs interacting with
DNA must stabilize their bond to the double helix by
creating a solid structure around it NAPs would
greatly strengthen the alignment along the DNA
longi-tudinal axis through the formation of hydrogen bonds
between phosphate groups of adjacent molecules
(Fig 4D,E) According to this model, NAPs would
thereby form a supra-molecular tunnel capable of
enveloping the entire DNA The final effect would be
the formation of an external scaffolding that protects the DNA by masking the sugar-phosphate backbone,
as indicated by the evidence of protection against DNase I and exonuclease III [29]
An additional property, namely conformational, might be ascribed to the differences, in terms of size
Fig 3 Structural models of nuclear aggregates of polyamines
(NAPs) (A) Small-size NAP (s-NAP) In accordance with the
simp-lest formula indicating a spermine (Sm) ⁄ spermidine (Sd) ⁄ putrescine
(P) ratio of 2 : 1 : 1, polyamines were represented as terminally
linked by phosphate groups to form a single cyclical structure (grey
disks, carbon atoms; blue disks, nitrogen atoms; yellow disks,
phosphate atoms; red disks, oxygen atoms; white disks, hydrogen
atoms) (B) Medium-size NAP (m-NAP) This NAP is represented as
a polymer of five s-NAPs linked by hydrogen bonds (green
tri-angles) The white arrows indicate the possible opening⁄ closure
movements that allow the adaptation of NAPs to DNA grooves.
The closure of the arch (resting state) may occur when the
com-pound is in phosphate buffer solution (C) Large-size NAP (l-NAP).
This NAP is represented as a polymer of five 6-polyamine units,
linked by hydrogen bonds, according to the simplest formula
indica-ting an Sm⁄ Sd ⁄ P ratio of 1 : 1 : 1.
Trang 8and shape, among the single NAPs Previously, we
have shown that m-NAP can enhance the
electropho-retic mobility of genomic DNA [29] Furthermore, we
showed by spectrophotometry that m-NAP has the
property to increase the absorbance at 260 nm of
genomic DNA, whereas other NAPs failed to do so
These results suggest that the m-NAP interacting with
DNA might determine structural rearrangements
char-acterized by base extrusion, an event occurring in the
transition to the left-oriented conformation [16] For
this reason, we predicted a model in which m-NAP
favours DNA transition to the Z-form (Fig 4C), a
DNA form characterized by a narrower diameter, more
elongated strands and outward exposure of bases
Our previous experiments suggest that the s-NAP
works both as a functional NAP, binding the DNA as
such, and as a precursor of the m-NAP Although the
aggregation of s-NAPs can occur independently of the
DNA structure, the possibility exists that m-NAPs
can be built up directly in loco, through the sequential
aggregation of three small units, to two s-NAPs
already bound to DNA Thereby, the progressive
for-mation of compounds with an increasing number of
monomers (up to five) would force, with a type of
wedge-like progression (Fig 4B), the DNA grooves to
widen and simultaneously determine the transition
towards the Z-form [16], which then proceeds along the two strands in a zip-like manner Another import-ant consequence of s-NAP complexation into m-NAP
is the strong increase of electrostatic forces that the latter compound exerts on DNA It is known that elec-trostatic forces play a greater role in the A–Z trans-ition than they do in the B–Z transtrans-ition, because the difference in the linear charges density is greater between the A and the Z forms than between the B and the Z forms [51] For this reason, we constructed
a model in which s-NAP interacts with A-DNA and, among the non-Zfi Z transitional possibilities, we chose the A fi Z possibility (Fig 4A,C) Additionally, because the A-DNA major groove is narrower than the B-DNA major groove, we hypothesized, by virtue
of size compatibility criteria, a preferential interaction
of the s-NAP with A-DNA (Fig 4A) In fact, we eval-uated the diameters of the monomers to be 15 A˚ for s-NAP and m-NAP and 25 A˚ for l-NAP
We do not have clear evidence for proposing an interactive model for l-NAP with a specific DNA type However, as l-NAP is the most widely represented compound in the nuclei of quiescent and replicating cells, and the B-DNA is the most common DNA form [52], a specific role for l-NAP in the protection and conformation of B-DNA can be suggested However,
Fig 4 Interaction of single nuclear aggre-gates of polyamines (NAPs) with different DNA forms (A) Small-size NAP (s-NAP) interacting with A-DNA Grey rings repre-sent the polyamine backbone Red dots indicate the phosphate groups To simplify interpretation of the figures, the NAPs phosphates facing the DNA groove were omitted A-DNA has a groove width more suitable than other DNA forms for interac-tion with this NAP (B) Progressive forma-tion of medium-size NAP (m-NAP) The addition of s-NAP units to two s-NAPs already bound to DNA (up to five) can allow the formation of m-NAP directly onto the DNA This may favour the transition to the Z-DNA, through the progressive widening of DNA strands and the exposure of bases (C) m-NAP interaction with Z-DNA Z-DNA sta-bilization by the m-NAP arch-like structure was represented as a result of the distan-cing of consecutive A-DNA major grooves (D) Perspective view of s-NAPs connected
by hydrogen bonds Aggregation of more s-NAPs units can allow the formation of a tunnel-like envelope around the DNA (E) Perspective view of m-NAPs connected by hydrogen bonds A 3D m-NAP tunnel structure enveloping the DNA is suggested.
Trang 9it should be mentioned that whatever the DNA
con-formation, DNA protection is fully assured by each
interacting NAP
Studies conducted (based on CD and⁄ or Raman
spectroscopy), to date, on the interaction between
polyamines and DNA, analysed DNA molecules much
smaller than the genomic DNA used in the present
study, testing polyamine⁄ DNA ratios of 1 : 10 to 1 : 1,
which was quite different from those used in our
experimental setting (NAP⁄ DNA ratio of 1 : 5000)
[1,50,53] For this reason, we are convinced that these
methodological approaches cannot be easily
extrapola-ted to the NAPs setting Therefore, much work must
be carried out to clarify several aspects of our model
However, a recent review from Medina and coworkers
stated that ‘despite the great amount of experimental
and theoretical works carried out up to now, it is not
possible to give an undoubted explanation about how
the polyamines bind to DNA’ [54] In this context, we
believe that our work, although not fully exhaustive in
all the aspects approached, might shed novel light on
the matter
In conclusion, we demonstrated that NAPs are able
to preserve the genomic DNA from DNase I-dependent
degradation, with an efficacy extraordinarily greater
than single polyamines Furthermore, we showed that
DNA, while preserved in its integrity by single NAPs,
undergoes temperature-sensitive conformational
chan-ges, which are indicative of a preserved DNA elasticity
We believe that NAPs are the sole biologically active
forms by which polyamines physiologically interact with
and protect genomic DNA, given that these quasi-stable
molecular aggregates, natural examples of
supramolecu-lar chemistry, are able to reach the maximum effect with
the minimum effort
Experimental procedures
Human genomic DNA was isolated from peripheral blood
leukocytes donated by M di Pietro DNA was extracted
and purified in phenol⁄ chloroform and then resuspended in
Tris⁄ EDTA (TE) buffer NAPs were extracted from the
nuclei of preconfluent Caco-2 cells and purified by gel
per-meation chromatography, as previously described [29]
Genomic DNA (4 lg per 2.5 lL of phosphate buffer)
was incubated for 6 min at 37C with 4.5 lL of l-, m- or
s-NAP (mean polyamine concentration: 0.25 ngÆlg)1DNA)
or water solutions of single polyamines (putrescine,
sper-midine and spermine) at a concentration of 1, 50, 150 or
600 lm The mixture was then exposed to DNase I
(RQ1RNase-free DNase; Promega, Milan, Italy) at a
con-centration of 0.025 UÆlg)1 DNA Briefly, 1 lL of the
DNase I solution was added to 1 lL of the reaction buffer
solution (400 mm Tris⁄ HCl, pH 8, 100 mm MgSO4 and
10 mm CaCl2) and then mixed with NAP–DNA or poly-amine–DNA solutions The enzyme action was stopped after 30 min of incubation at 37C by adding 1 lL of
20 mm EDTA, pH 8 Samples were then loaded onto a 1Æ5% (w⁄ v) ultrapure DNA grade agarose gel Electrophor-esis of DNA was carried out for 1 h in an HE 100 supersub (Amersham Pharmacia Biotech, Uppsala, Sweden), at a constant temperature of 37 or 40C (controlled by a peri-staltic pump system), by applying an electric field strength
of 11.1 VÆcm)1in Tris⁄ borate ⁄ EDTA buffer Each gel was then photographed by using a Polaroid MP-4 L camera NAPs modelling was carried out, producing molecular structures that were in strict accordance with biochemical data and theoretical rationales
All biochemical data were collected from analytical, elec-trophoretic and NMR studies shown in our previously pub-lished work [29] and in the present study Theoretical rationales were derived from the universally accepted prin-ciples of the supramolecular chemistry based on the self-assembly by means of weak interactions [47,55–57] A multistep aggregation plan was developed as follows (a) a cyclic and planar structure representing the single base module, (b) base modules combined in polycyclic planar structures (ladder), (c) the polycyclic modules developed 3D into tunnel-like structures The compatibility between the NAPs dimensions, evaluated on formulas, and the currently accepted DNA grooves size was the requisite for the inter-action scheme of NAPs with DNA forms
Acknowledgements
We are grateful to Dr Alma Contegiacomo and Dr Luigi Gomez-Paloma for helpful suggestions and advice, to Mr Paolo Mastranzo for technical assistance and to Mr Stefano D’Agostino for his contribution in artwork production This work was supported by a research grant from the Campania Region
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