Urea Mimics Nucleobases by Preserving the Helical Integrity of BDNA Duplexes via Hydrogen Bonding and Stacking Interactions
Thesis submitted in partial fulfillment of
the requirements for the degree of
Master of Science by Research
in Computational Natural Science
By
Indrajit Patil
200964016
[email protected]
INTERNATIONAL INSTITUTE OF INFORMATION TECHNOLOGY
(Deemed to be University)
Hyderabad – 500032, India
July, 2016
Copyright © Indrajit Patil, 2016
All Rights Reserved
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ACKNOWLEDGEMENT
It is my great pleasure to acknowledge the many people who have helped
me during my stay here at IIIT.
First and foremost, I would like to thank my guide, Dr. U Deva
Priyakumar. Sir has been an inspiration for me throughout my stay here and a wonderful guide
during my thesis. He patiently addressed all my queries and provided constant support when I
needed it the most. I have learnt so many things from him and would try and emulate his very
qualities in my life ahead.
I would also like to thank all the CCNSB faculty for their constant support
and patience during our course work. I would also like to thank all the faculty here at IIIT
Hyderabad. In hindsight, the quality of education imparted here is unparalleled and of the highest
standard. I would always cherish and respect all the faculty for shaping us into what we are
today. They would have a very big role in whatever we achieve in our respective careers ahead.
I would like to thank all my lab seniors for helping me and guiding me
during the course of my thesis. Suresh sir, Siladitya sir, Pratyusha mam, Koushik sir, Gayathri,
Shwetha mam, Sandhya mam, Ramki sir, Ragini, Antarip sir who have supported and guided me
at every point.
I would also like to thank all my batchmates for making the stay a very
memorable one. There have been a lot of fun memories which will last a lifetime. Also, the
healthy competition and quality of assignments have helped shape our characters and
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personalities. I would also like to thank Kuldeep and Supriya for motivating me and supporting
me during the course of my thesis.
I would also like to thank Mr. Girish Byrappa and Mr. Balsantosh the
CCNSB administrative assistants for providing assistance. I would also like to thank Mr. Umesh
for maintaining an academic atmosphere in the lab by keeping it clean at all times.
I would also like to thank my parents for making me whatever I am today.
My parents taught me and sacrificed so many things to see me succeed in life. I hope I am able to
live up to their expectations and take care of them.
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ABSTRACT
One of the DNA lesions formed due to free radical damage of thymine is
urea, occurrence of which in DNA blocks DNA polymerases, and has been shown to be lethal.
Recently, it has been shown that urea is capable of forming hydrogen bonding and stacking
interactions with nucleobases, which are responsible for the unfolding of RNA in aqueous urea.
Base pairing and stacking are inherent properties of nucleobases; since urea is able to form both,
this study attempts to investigate if urea can mimic nucleobases in the context of the nucleic acid
structures by examining the effect of introducing urea lesions complementary to the four
different nucleobases on the overall helical integrity of B-DNA duplexes and their
thermodynamic stabilities using molecular dynamics (MD) simulations. The MD simulations
resulted in stable duplexes without significant changes in the global B-DNA conformation. In
agreement with experimental results, the urea lesions occupy intrahelical positions by forming
hydrogen bonds with nitrogenous nucleobases. Further, these urea lesions form hydrogen
bonding and stacking interactions with other nucleobases of the same and partner strands, which
are sometimes similar to the nucleobases in typical B-DNA duplexes. Direct hydrogen bond
interactions are observed for the urea-purine pairs within DNA duplexes, whereas two different
modes of interactions, namely direct hydrogen bonds and water-mediated hydrogen bonds, are
observed for the urea-pyrimidine pairs. The latter explains the complexities involved in
interpreting the experimental NMR data reported previously. Binding free energy calculations
were further performed to understand the thermodynamic stability of the urea incorporated DNA
duplexes with respect to pure duplexes. This study suggests that urea potentially mimics
nucleobases by pairing opposite to all the four nucleobases and maintains the overall structure of
the B-DNA duplexes.
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TABLE OF CONTENTS
CHAPTER 1: Introduction ........................................................................................................17
CHAPTER 2: Computational Methods ....................................................................................20
2.1 Quantum Mechanical Calculations .............................................................................20
2.2 Molecular Dynamics Simulations ...............................................................................21
2.3 Free Energy Calculations ............................................................................................24
CHAPTER 3: Results and Discussions .....................................................................................25
3.1 QM calculations on urea-nucleobase pairs .................................................................25
3.2 Stable structures obtained from initial simulations ....................................................26
3.3 Discussions based on long MD simulations ..............................................................38
CHAPTER 4: Conclusions ........................................................................................................57
References ..................................................................................................................................61
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LIST OF FIGURES
Figure 2.1.1: Model systems of urea-nucleobase pairs and DNA duplexes. (a-g) Representation
of the possible hydrogen bonding between urea and all the four nucleobases (A, G, T and C), and
(h) the DNA sequence used to model the pure and mismatched B-DNA duplexes.
......................................................................................................................................................22
Figure 3.1.1: Various conformations depicting hydrogen bonding between urea and the
nucleobases . The interaction energies for each of the conformations are also given. ................25
Figure 3.1.2: Average RMSD values for all the urea incorporated DNA duplexes averaged over
the simulation period ....................................................................................................................27
Figure 3.1.3 : Conformations changing to other conformations within a few ns of MD
simulations. ..................................................................................................................................28
Figure 3.1.4: Times series of root mean square (RMS) deviations for all the duplexes in
reference to their respective initial structure. ..............................................................................29
Figure 3.1.5: Probability distribution of the two hydrogen bonds between urea and the
complementary bases present in all the DNA duplexes. .............................................................30
Figure 3.1.6: Probability distribution of all base pairs in the double helix except urea-base
pair................................................................................................................................................32
Figure 3.1.7: Probability distributions of backbone alpha angles corresponding to urea and its
complementary nucleotides in all the stable urea incorporated DNA duplexes. .........................33
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Figure 3.1.8: Probability distributions of backbone beta angles corresponding to urea and its
complementary nucleotides in all the stable urea incorporated DNA duplexes. .........................34
Figure 3.1.9: Probability distributions of backbone epsilon angles corresponding to urea and its
complementary nucleotides in all the stable urea incorporated DNA duplexes. .........................35
Figure 3.1.10: Probability distributions of backbone gamma angles corresponding to urea and its
complementary nucleotides in all the stable urea incorporated DNA duplexes. .........................36
Figure 3.1.11: Probability distributions of backbone zeta angles corresponding to urea and its
complementary nucleotides in all the stable urea incorporated DNA duplexes. .........................37
Figure 3.2.1: Time series of root mean square deviations (in Å) of the urea incorporated B-DNA
duplexes. ......................................................................................................................................39
Figure 3.2.2: Probability distributions of distances (in Å) corresponding to the hydrogen bonds
between urea and all the four nucleobases that are present in urea incorporated B-DNA duplexes.
......................................................................................................................................................41
Figure 3.2.3: Probability distributions of hydrogen bond distances (in Å) of all the base pairs in
the urea incorporated B-DNA duplexes during the MD simulations. .........................................42
Figure 3.2.4: : Interaction energies of the urea-nucleobase mismatched pairs and their neighbour
AT and GC base pairs of all the urea incorporated B-DNA duplexes (blue). The interaction
energies corresponding to base pairs in pure DNA are also included for comparison (red). ......44
Figure 3.2.5: : Probability distribution of the distances (in Å) between connecting nitrogen
atoms of nucleobase or urea with furanose sugar for (a) urea-nucleobase mismatched pairs and
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(b) base pairs next to the mismatched base pair (CG:red and AT:black) present in all the urea
incorporated B-DNA duplexes. ..................................................................................................46
Figure 3.2.6: Two modes of interactions between urea and pyrimidine bases. Depiction of direct
hydrogen bonds and water mediated hydrogen bonds of urea with its complementary bases,
thymine (a:T2c) and cytosine (b:C2b), during the course of the molecular dynamics simulations.
......................................................................................................................................................47
Figure 3.2.7: Probability distributions of pseudorotation angles of furanose sugars of urea
nucleotides (Ur) and their respective complementary nucleotides (cU) in all the urea incorporated
B-DNA duplexes. The probabilities corresponding to Ur and cU are shown in blue and green
respectively, while the probability corresponding to pure DNA duplexes is shown in purple. ...49
Figure 3.2.8: Probability distributions of pseudorotation angles of furanose sugars for the (a)
full DNA duplex, (b) full DNA duplex without urea-nucleobase pair, and (c, d) neighbour base
pairs in all the urea incorporated DNA duplexes. The pseudorotation angles corresponding to the
furanose sugars of typical B-DNA duplexes are shown in black while the urea incorporated DNA
duplexes are shown in red. ............................................................................................................51
Figure 3.2.9: Probability distributions of sugar-base glycosidic angle (in degree) for urea
nucleotides (a) and respective complementary nucleobases (b) of all the urea incorporated BDNA duplexes. ..............................................................................................................................52
Figure 3.2.10: Probability distributions of backbone dihedral angles (in degree) corresponding to
the urea nucleotides, its complementary nucleotide and their neighbour nucleotides in all the urea
incorporated DNA duplexes. ........................................................................................................53
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Figure 3.2.11: Probability distributions of phosphodiester bond angle (in degree) corresponding
to the urea nucleotides, its complementary base and their neighbour nucleotides in all the urea
incorporated DNA duplexes. .......................................................................................................54
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LIST OF TABLES
Table 3.2.1 : Stacking interaction energies of urea and its complementary nucleobases with the
nucleobases of the same and their complementary strands. All the energies are in kcal mol-1. ..45
Table 3.2.2: Number of water molecules present around the mismatched base pair (urea and
complementary base) for all the urea incorporated B-DNA duplexes considered in the present
study. .............................................................................................................................................48
Table 3.2.3: Absolute (ΔG) and relative (ΔΔG) binding free energy values calculated for the
duplex formation of B-DNA duplexes considered in the present study. The relative binding free
energy values are calculated with respect to their pure DNA duplex. All the energy values are in
kcal mol-1. ....................................................................................................................................55
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Chapter 1
INTRODUCTION
Many exogenous and endogenous agents are capable of damaging cellular DNA,
termed as DNA damage, thereby modifying the properties of DNA or triggering mutations. Such
modifications have been shown to be responsible for mutagenesis, carcinogenesis and cell
lethality [1]. DNA damage includes base and furanose sugar modifications, abasic site formation,
single and double strand breaks, and cross linkages [1-3], and these factors play a central role in
the etiology of cancer, neurological disorders and aging related diseases [4-8]. One of the most
common DNA damages is the formation of thymine glycol (Tg) from thymine upon oxidative
addition. Thymine glycol undergoes further ring fragmentation to form N-(2-deoxy-β-D-erythropentofuranosyl) formamide and N-(2-deoxy-β-D-erythro-pentofuranosyl) urea [9-13]. These
fragmented products are considered as intermediate structures between nucleobases and abasic
sites. These urea lesions were reported as being non-instructive [14, 15] and are known to be
mutagenic, similar to abasic sites [16]. Previous studies have shown that urea lesions are capable
of forming hydrogen bonds with nucleobases after losing part of their original coding
information [9, 10]. Urea lesions are poorly bypassed by DNA polymerase and they block DNA
polymerase in in vitro [15, 17]. These urea lesions are recognized by enzymes like endonuclease
III, which are known to remove the lesions in Escherichia coli [18]. If these modifications are
not repaired, the polymerase can potentially incorporate any base opposite to it, which results in
a mutation. It has been observed that the presence of urea and Tg affects the cleavage rate of
RNA-DNA hybrids by ribonuclease H, and it preferentially redirects the cleavage site lying next
to the mismatch base pair [19]. Previous studies have also shown that the damaged site increases
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the flexibility of duplex DNA that increases protein recognition of DNA during DNA repair
mechanisms [20].
NMR studies have been performed on the B-DNA duplexes containing urea or
formamide lesion as a bulge or complementary to the nucleobases [21-29]. NMR studies of DNA
with urea lesion opposite to the thymine base have indicated that the urea nucleotide adopts cis
and trans conformations around the uridic bond, and these two isomers were present in the ratio
of 2:3 in solution [23]. It was also observed that urea-thymine base pair has two hydrogen bonds
similar to regular AT base pair [23]. Furthermore, the mismatched base pair occupied an
intrahelical position and was held by hydrogen bonds with other nucleobases. NMR studies have
also reported the structures of B-DNA helix with urea lesion as a bulge opposite to the abasic
site, and explained their role in frameshift mutagenesis [26]. This study had also suggested that a
kink was produced in the double helix due to the formation of hydrogen bonds with the
nucleobases in the same strand [26]. During replication, if the polymerase does not include a
nucleobase opposite to the urea lesion, then it creates frameshift mutagenesis. Such a structure
was reported in previous studies [27]. But these studies were unable to characterize the
individual structures of DNA duplexes with urea lesion in cis and trans isomers due to resonance
overlap. Unlike formamide, the urea lesion occupies intrahelical and extrahelical positions
because of the capability of urea lesion to form unusual hydrogen bonds. Similarly, apurinic and
apyrimidinic abasic sites in DNA duplexes occupy both intrahelical and extrahelical
conformations as observed in several experimental studies [30-35].
Recently we have investigated the nonbonded interactions that are responsible for the
unfolding of RNA in aqueous urea [36]. It has been shown that urea is capable of forming
hydrogen bonding interactions and more interestingly stacking interactions with nucleobases,
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which is responsible for stabilizing the bases in their extrahelical conformation and hence
favoring the unfolded states, which is otherwise not possible in presence of only water [36-38].
Hydrogen bonding and stacking interactions between nucleobases are two important phenomena
that are responsible for the stability of the duplex structures of DNA. Since urea is capable of
forming both, the natural question is how well urea can mimic the nucleobases within the nucleic
acid structures. Given that urea is a thymine damaged product and that it is present in DNA
structures, this question assumes higher significance in terms of understanding the structures,
dynamics, and thermodynamic stabilities of nucleic acid duplexes containing urea lesions.
Although NMR experiments provide valuable information about the structures of urea and
formamide incorporated B-DNA duplexes, the complete characterization of DNA duplexes with
urea lesions has been difficult because of the resonance overlapping of the signals [23, 26].
Molecular dynamics (MD) simulations are, in general, very useful in studying the structure,
dynamics and thermodynamic stability of damaged and chemically modified nucleic acid
duplexes [39-42]. In the present study, MD simulations have been performed on the B-DNA
duplexes with urea lesion opposite to four nucleobases adenine (A), thymine (T), guanine (G),
and cytosine (C) in Watson-Crick (WC), Hoogsteen, and sugar edged interactions. Analyses of
the MD trajectories suggest that the urea lesions prefer to form WC-like hydrogen bond
interactions with the nucleobases, especially purines and can potentially mimic the nucleobases
in B-DNA duplexes.
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Chapter 2
COMPUTATIONAL METHODS
2.1 Quantum Mechanical Calculations
To investigate the paring nature of urea with nitrogeneous bases, all the canonical and
noncanonical orientations like Watson-Crick and Hoogsteen etc were considered between urea
and nitrogeneous bases. The initial geometries corresponding to pairs of urea and nucleobases A,
G, C, T were generated using GaussView 05 [43] software. All these geometries were optimized
using aug-cc-pVDZ basis set
at MP2 level of theory with resolution of identity (RI)
approximation (RI-MP2) in TURBOMOLE 6.2 program [44] and the interaction energies were
corrected according to Basis set super position errors (BSSE) method using RI-MP2 level of
theory in GAMESS [45] software. The interaction energies were calculated as the difference
between energies of urea-nucleobase pair and sum of urea and nucleobase energies. All the ureanucleobase binary complexes from this study were considered for generating the initial
configurations of the urea incorporated DNA duplexes. Based on the structures of typical DNA
duplex structures, it is known that the distance between nitrogen atoms (N9 for purines and N1
for pyrimidines) connected to the furanose sugars of the nucleobases from the two
complementary strands is approximately 9 Å. Based on this, a cut-off distance of 9 ± 2 Å
between either of the N atom of urea and N9 of purines or N1 of pyrimidines was used to select
urea-nucleobase pairs that are suitable to fit within the B-DNA duplex (Figure 2.1.1a-g).
Initial models for urea incorporated DNA duplexes: This selection criterion resulted in
thirteen different structures of urea-nucleobase pairs with WC, Hoogsteen and sugar edge-like
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interactions. The 10-mer DNA sequence from the available NMR experimental study on ureathymine base pair [23] was considered as the template sequence for modeling all the pure and
urea incorporated B-DNA duplexes (Figure 2.1.1h). The thymine-urea pair present in this
structure was replaced by each of the urea-nucleobase pairs resulting in thirteen duplex structures
containing the urea lesion (Figure 2.1.1a-g). Corresponding DNA duplexes with the typical GC,
CG, AT and TA base pairs were also considered for comparison. All the urea modifications were
done by using the SYBYL program (Tripos Inc).
2.2 Molecular dynamics simulations:
CHARMM bimolecular simulation program [46] was used to prepare the initial systems of urea
incorporated B-DNA duplexes, to setup the MD simulations, to equilibrate the systems and to
perform analyses of the MD trajectories. All the production simulations were performed by
employing the NAMD biomolecular simulation program [47]. CHARMM36 all-atom parameters
[42, 48-50] were used for B-DNA duplex and urea. The topology and parameters corresponding
to urea nucleotide were derived from the CHARMM Generalized Force Field (CGenFF) by
combining urea, nucleotide and amide parameters [51]. 500-step minimization was performed on
each duplex using the steepest descent (SD) method by applying NOE constraints on the
hydrogen bonds present between urea and its complementary base in all the B-DNA duplexes.
Another 500-step SD minimization was performed on these systems with harmonic restraints on
the heavy atoms. Then these systems were overlaid into an orthorhombic waterbox whose
dimensions were selected by extending 10 Å beyond the duplex dimensions. Modified TIP3P
water model [52] was used for water.
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Figure 2.1.1: Model systems of urea-nucleobase pairs and DNA duplexes. (a-g) Representation of the
possible hydrogen bonding between urea and all the four nucleobases (A, G, T and C), and (h) the DNA
sequence used to model the pure and mismatched B-DNA duplexes. This sequence is taken from an
earlier NMR spectroscopic study on 10-mer DNA duplex with urea-thymine mismatch pair [21]. The
terms used for representing the urea incorporated DNA duplexes and throughout the thesis are also
mentioned.
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The negative charge of the phosphate groups of DNA duplexes were neutralized by
adding sodium ions, and then the systems were minimized by performing 500 steps each of SD
and adopted basis Newton Raphson (ABNR) minimizations while harmonically restraining the
DNA heavy atoms. A 100 ps MD simulation was performed on each of these systems in NVT
ensemble under the same conditions used above. The final coordinates obtained after the
equilibration were then used for running production simulations in NPT ensemble. SHAKE
algorithm [53] was used to constrain the covalent bonds involving hydrogen atoms and
CRYSTAL module [54] in CHARMM was used to represent periodic boundary conditions
during equilibration. Particle mesh Ewald (PME) summation method [55, 56] was used to treat
the long range electrostatic interactions. Lennard-Jones (LJ) potential was truncated at 14 Å by
using a force switch function [57]. An integration time step of 2 fs was used and the coordinates
were saved every 5 ps for further analysis. All the urea incorporated DNA duplexes were
simulated for 25 ns without any restraints for further equilibration, which were considered as the
second stage of equilibration. Further, 100 ns MD simulations were performed on the duplexes in
which the urea lesions occupy intrahelical positions. The stable conformations obtained at the
end of the 25 ns MD simulations were used to run these simulations (see later). The temperature
and pressure were maintained using the Nosé-Hoover thermostat [58] and Langevin piston [59]
respectively during the simulations. Four additional simulations of duration 100 ns each were
performed on pure DNA duplexes corresponding to AT, GC, TA and CG base pairs for
comparison (Figure 2.1.1h). Visual Molecular Dynamics (VMD) program [60] was used for
viewing the structures and rendering the images shown in the manuscript.
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2.3 Free energy calculations:
The molecular mechanics-generalised Born with surface area (MM-GBSA) method was
used to calculate the binding free energies corresponding to formation of duplex from individual
strands which uses molecular mechanical energy, solvation energy and entropy of the system
[36, 37].
where
represents the free energy of the system,
represents the molecular mechanical
energy which is the sum of all the energy terms in the force field equation,
is the solvation
free energy which is the sum of electrostatic (
) solvation free
energies,
) and non-polar (
is the absolute temperature and
is the molecular mechanical entropy of the
system. The electrostatic and non-polar solvation free energies were calculated by using the
Generalised-Born molecular volume (GBMV) method and
0.0072 kcal mol-1Å-2 respectively. The
, where
=
represents the solvent accessible surface area
calculated by using a small probe with radius 1.4 Å. The binding free energy can be calculated as
the difference in the free energies of the duplex, and the sum of the free energies of the
individual components.
where
represent the free energies of the duplex and individual
strands present in the duplex calculated using the above equation.
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Chapter 3
RESULTS AND DISCUSSION
3.1 QM calculations on urea-nucleobase pairs
Figure 3.1.1: Various conformations depicting hydrogen bonding between urea and the
nucleobases . The interaction energies for each of the conformations in kcal/mol are also given.
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We have performed QM calculations to investigate all possible hydrogen bonded
interactions of urea with all the DNA nucleobases and have observed that such hydrogen bonded
interactions give rise to stabilization energies in the range of −6 to −21 kcal/mol (Figure 3.1.1).
The interaction energies of nucleobases and urea calculated from the above study are comparable
to the interaction energies of the usual base pairs. We can thus conclude that urea forms strong
hydrogen bonded interactions with all the nucleobases in many of the conformations. To study
how urea would interact with nucleobases inside the DNA double helix we introduce a
mismatched base pair with urea replacing a nucleobase in one of the strands. Using the selection
criteria based on the cut-off distance of 9 ± 2 Å between either of the N atom of urea and N9 of
purines or N1 of pyrimidines described above, we modeled the mismatched base pair according
to the conformations studied in the QM calculations and performed molecular dynamics
simulations to study the stability of the double helix which incorporates the mismatched base
pair in various conformations.
3.2 Stable structures obtained from initial simulations
Structural Deviations and Dynamics:
The propagation of urea incorporated DNA duplexes are assessed by calculating root mean
square (rms) deviations. The rms deviations corresponding to all the 13 urea incorporated
systems were calculated with respect to their starting conformations along the simulation time
(Figure 3.1.2). The rms deviations corresponding to bases and backbone of the duplexes were
also calculated . The variations in rms deviations of full duplex, bases and backbone indicate that
the structural deviations are small compared to their respective starting structure, indicating close
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behaviour with the initial conformation. Visual inspection of the trajectories showed that the
DNA duplex with C2c conformation lost its base pairing interactions and became unstable. This
can be seen by the higher rms deviation values corresponding to the duplex with conformation
C2c than the other duplexes (Figure 3.1.2).
Figure 3.1.2: Average RMSD values for all the urea incorporated DNA duplexes averaged over
the simulation period.
Thus, this conformation, C2c, is not considered for further analysis. Similar to previous NMR
studies, all the urea nucleotides occupied intrahelical position in the duplex. Interestingly, some
of the urea-nucleobase pair conformations simply transformed into other conformations within a
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few ns of the simulation time, indicating the stability of the duplexes with certain base pair
conformations. The duplexes with conformations A3b, A3c, A6b and A6c converted to other
conformations A3a, A5c, A2b and A2c respectively (Figure 3.1.3).
Figure 3.1.3: Conformations changing to other conformations within a few ns of MD
simulations.
So the further analysis is restricted to the duplexes with stable conformations only. Hence the
total number of final structures considered for further analysis were reduced from 13 to 8.
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To understand the similarity of the urea incorporated DNA duplexes with typical B-DNA
structure, rms deviations with respect to the typical B-DNA duplex conformation were also
calculated for all the duplexes. The time series of rmsd plots indicate that the urea incorporated
duplexes do not deviate much from the typical B-DNA conformation, indicating the preservation
of B-form characteristics of the urea modified duplexes (Figure 3.1.4). The small deviations
observed in both backbone and base of all these stable duplexes further support the B-form
nature of the duplexes.
Figure 3.1.4: Times series of root mean square (RMS) deviations for all the duplexes in
reference to their respective initial structure.
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Figure 3.1.5: Probability distribution of the two hydrogen bonds between urea and the
complementary bases present in all the DNA duplexes.
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Hydrogen Bonds Between Urea and Nucleobases:
The initial structures have two hydrogen bonds between urea and nucleobases (Figure 3.1.1).
The time series plots corresponding to these two hydrogen bonds were calculated for all the ureanucleobase pairs present in the urea incorporated duplexes studied here (Figure 3.1.5).
As shown in Figure 3.1.5, the two hydrogen bonds between urea and cytosine
paired in C2c conformation are not stable as seen from the high bond distances (>4.5 Å),
indicating instability of the duplex in this conformation. In support with the rmsd values, the
variations observed for the distances corresponding to the two hydrogen bonds present in A3b,
A3c, A6b, and A6c structures indicate their transformation into other conformations. The
remaining structures have at least one stable hydrogen bond throughout the simulation,
indicating the stability of the urea-nucleobase base pairs. It is also noticed that frequent
formations and breakages of the hydrogen bonds are observed in urea-pyrimidine base pairs
while such kind of transformations are not observed for urea-purine base pairs. It is seen
throughout the simulation time that the hydrogen bonds between the remaining base pairs in the
double helix are not affected and are like the purine-pyrimidine base pairing in the usual DNA
double helix as shown by the probability distributions in the Figure 3.1.6.
Conformation of the Urea Nucleotide Sugar and Backbone:
The effect of the urea nucleotides on the conformational properties of the DNA duplexes were
examined in terms of pseudorotation angles and backbone dihedral angles such as α, β, χ, ε, γ, δ .
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Figure 3.1.6: Probability distribution of all base pairs in the double helix except urea-base pair.
The deoxyribose sugars generally show high populations in C2'-endo and small populations in
O4'-endo regions. But the incorporated urea strongly influences this ratio and increases
populations in C2'-endo region. Considerable changes are not seen in the conformation of the
furanose sugars of bases complementary to the urea nucleotides.
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Figure 3.1.7: Probability distributions of backbone alpha angles corresponding to urea and its
complementary nucleotides in all the stable urea incorporated DNA duplexes. Here the Nc - Base
complementary to urea, N1b - Base above Nc, N2b - Base below Nc, N1a - Base above Urea,
and N2a - Base below urea
The urea incorporation does not induce considerable changes in the backbone
conformation revealed by the probability distributions of the characteristic backbone dihedral
angles corresponding to urea nucleotide and its neighbour nucleotides (Figures 3.1.7-3.1.11).
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Figure 3.1.8: Probability distributions of backbone beta angles corresponding to urea and its
complementary nucleotides in all the stable urea incorporated DNA duplexes. Here the Nc - Base
complementary to urea, N1b - Base above Nc, N2b - Base below Nc, N1a - Base above Urea,
and N2a - Base below urea
34
Figure 3.1.9: Probability distributions of backbone epsilon angles corresponding to urea and its
complementary nucleotides in all the stable urea incorporated DNA duplexes. Here the Nc - Base
complementary to urea, N1b - Base above Nc, N2b - Base below Nc, N1a - Base above Urea,
and N2a - Base below urea
35
Figure 3.1.10: Probability distributions of backbone gamma angles corresponding to urea and its
complementary nucleotides in all the stable urea incorporated DNA duplexes. Here the Nc - Base
complementary to urea, N1b - Base above Nc, N2b - Base below Nc, N1a - Base above Urea,
and N2a - Base below urea
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Figure 3.1.11: Probability distributions of backbone zeta angles corresponding to urea and its
complementary nucleotides in all the stable urea incorporated DNA duplexes. Here the Nc - Base
complementary to urea, N1b - Base above Nc, N2b - Base below Nc, N1a - Base above Urea,
and N2a - Base below urea
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3.3 Discussions based on long MD simulations
The structural deviations of urea incorporated duplexes were assessed by
calculating root mean square deviations with respect to their initial conformation. Rmsd values
corresponding to all the eight urea incorporated systems were calculated with respect to their
starting conformations. The deviations were considerably small for all the duplexes within 25 ns
simulation time. Interestingly, the non-WC urea-nucleobase pairs either transformed into WC
conformations or simply move out of the duplex within few nanoseconds of the simulation time.
Elimination of the structures based on the stability and helical position of the urea nucleotides
decreased the number of structures under study from 8 to 5. These five structures are A2b, G1a,
G1c, C2b and T2c, on which a new set of simulations for 100 ns each were performed. The
current section describes the results of these five urea incorporated duplexes obtained from the
100 ns MD simulations.
Structure and dynamics of urea incorporated B-DNA duplexes:
The structural closeness of the damaged DNA duplexes was examined by calculating the rmsd
with respect to their initial conformation that resembles a typical B-form DNA. As shown in
Figure 3.2.1, the rmsd values indicate that the structural deviations are small compared to their
respective starting structure, indicating their similarity with an ideal B-form DNA. Visual
inspection of MD snapshots indicates that the urea lesions are placed well inside the duplex for
all the urea incorporated B-DNA during the course of these simulations.
Furthermore, the rmsd values were calculated with respect to typical B-DNA
conformation, and the small values observed indicate that the presence of urea does not affect
38
their overall structure significantly. This suggests that the B-DNA duplexes preserve their global
characteristics after the incorporation of urea lesions opposite to all the four nucleobases. High
deviations were observed for short time within these simulations, especially for T2c and C2b,
which are attributed to local structural transitions that are discussed later. The simulations study
resulted in stable duplexes that are similar to the structures observed in earlier NMR
spectroscopy studies on DNA duplex with urea complementary to thymine base [23].
Figure 3.2.1: Time series of root mean square deviations (in Å) of the urea incorporated B-DNA
duplexes. These values are calculated with respect to their initial conformation used to run the
simulations.
39
Hydrogen bonding interactions between urea and nucleobases:
Previous studies have shown that urea has strong capability to form hydrogen bonds with the
nucleobases [40-42]. There are two potential hydrogen bonding sites on the urea molecule to pair
with the nitrogenous bases of the complementary strand. These are oxygen atom on the carbonyl
group of urea and nitrogen atom of the amino group which is not attached to the deoxyribose
sugar of the double helix. In the beginning, all the urea-nucleobase pairs have two hydrogen
bonds between urea and nucleobases (Figure 2.1.1a-g). Stability of these hydrogen bonds was
monitored by calculating the hydrogen bond distances corresponding to the donor/acceptor
atoms involved in hydrogen bonding between urea and nucleobases during the simulations.
Figure 3.2.2 shows the probability distributions of such hydrogen bond distances for all the ureanucleobases pairs present in the urea incorporated duplexes. These distributions show interesting
trends that differentiate the urea-purine and urea-pyrimidine pairs. In the duplexes containing
urea-purine pairs, a strong peak at 3.5 Å suggests that at least one stable hydrogen bond is
observed during the simulations in spite of deviations in the second hydrogen bond (Figure
3.2.2). The probability distributions of G1c indicate the GUA-urea pair to be alternating between
two-hydrogen bonded state and a bifurcated hydrogen bonded state. In case of urea-pyrimidine
pairs, the peak at 3.5 Å corresponds to the direct hydrogen bond interactions between urea and
nucleobase, whereas the peak at distance 5.5 Å corresponds to water mediated hydrogen bond
formation between urea and nucleobase. This suggests that, while urea interacts with the purine
bases through direct hydrogen bonding interactions, urea-pyrimidine pairs alternate between
direct hydrogen bonding and water mediated interactions within the duplex. The nature of such
water mediated interaction between the pyrimidine nucleobase and urea are discussed later. The
base pairs other than the mismatched urea and its neighbors are reasonably stable with WC base
40
pairing (Figure 3.2.3) indicating that inclusion of urea lesion in the B-DNA duplexes does not
disturb their structural integrity.
Figure 3.2.2: Probability distributions of distances (in Å) corresponding to the hydrogen bonds
between urea and all the four nucleobases that are present in urea incorporated B-DNA duplexes.
The hydrogen bonding between urea and nucleobase are shown in a separate panel on the right
hand side. The color scheme used to represent probability distribution lines is same as the color
scheme of hydrogen bonds between the urea-nucleobase pairs.
41
Figure 3.2.3: Probability distributions of hydrogen bond distances (in Å) of all the base pairs in
the urea incorporated B-DNA duplexes during the MD simulations.
Energetics of the hydrogen bonding and stacking interactions between urea and
nucleobases:
Base pairing and stacking interactions among the nucleobases are important for the stability of
nucleic acid duplexes. The strength of these interactions in the presence of the urea lesion were
calculated and compared to the regular DNA duplexes. The mean interaction energies of urea
42
with its complementary WC base are shown in Figure 3.2.4. The interaction energy values in the
range from ~ -5 to -15 kcal/mol suggest that urea strongly interacts with all the nucleobases
though most of the energies are less favorable compared to those of the regular A-T and G-C
base pairs (~ -14 and -24 kcal/mol for the canonical AT and GC base pairs) of a typical B-DNA
duplex. The effect of the presence of urea on the strength of neighboring base pairs was also
evaluated (Figure 3.2.4), which indicate no significant change in the interaction energies due to
the inclusion of urea lesion. Though the interaction energies between the nucleobases and urea
are comparable or less favorable compared to canonical AT base pair, they are significant and
seem strong enough to stabilize these duplexes. Stacking interactions of urea moiety with the
nitrogenous bases present above and below in the same strand and complementary strand were
also calculated, and are shown in Table 3.2.1. The energy values indicate that the urea moiety
present in DNA duplexes forms significant stacking interactions with the neighboring bases.
However, similar to the hydrogen bonding interactions, the magnitudes of stacking interaction
energies of urea moiety are less favorable than that observed in the control simulations.
However, these interactions are sufficient to keep the urea lesions inside the double helix in a
manner characteristic of regular bases. Such strong stacking interactions between urea and
nucleobases were also observed in previous quantum chemical and MD simulation studies in the
context of RNA unfolding in presence of aqueous urea [36-38]. The incorporation of urea lesion
leads to small deviations in the stacking interaction energies of nucleobases complementary to
urea (Table 3.2.1). Therefore, the energy values from hydrogen bond interactions and stacking
interactions though less favorable than in canonical DNA duplexes suggest that urea can
potentially replace nucleobases in DNA duplexes without distorting their overall structures. The
43
effect of incorporating a urea lesion on the overall stability of the duplex structures is discussed
later.
Figure 3.2.4: Interaction energies of the urea-nucleobase mismatched pairs and their neighbour
AT and GC base pairs of all the urea incorporated B-DNA duplexes (blue). The interaction
energies corresponding to base pairs in pure DNA are also included for comparison (red). All the
interaction energies are in kcal mol-1.
Smaller size of urea compared to the purine base leads to interesting hydrogen bond
dynamics:
All canonical base pairs in nucleic acids are formed between a purine and a pyrimidine
nucleobase.
44
Table 3.2.1: Stacking interaction energies of urea and its complementary nucleobases with the
nucleobases of the same and their complementary strands. All the energies are in kcal mol-1.
Duplex
B-DNA
Urea
Complementary
-19.0 ± 0.0
-19.5 ± 0.0
-5.6 ± 0.0
-19.4 ± 0.0
-18.5 ± 0.3
-20.8 ± 0.0
G1a
-5.6 ± 0.0
-21.6 ± 0.1
G1c
-7.3 ± 0.0
-20.4 ± 0.0
-12.0 ± 0.0
-9.6 ± 0.0
-6.4 ± 0.1
-9.9 ± 0.1
-13.9 ± 0.0
-8.9 ± 0.1
-5.2 ± 0.1
-10.8 ± 0.2
A2b
B-DNA
B-DNA
T2c
B-DNA
C2b
In the duplexes considered here, one of the nucleobases is replaced by the urea moiety. While the
size of the urea moiety and the positions of the hydrogen bonding groups are comparable to those
of the pyrimidine base, there exists a huge mismatch in terms of the size when it comes to
replacing a purine base by urea. As discussed above, at least one conventional hydrogen bond is
formed between the pairs involving urea and GUA/ADE. In case of urea-CYT/THY pairs, the
sugar moieties have to move towards each other to form hydrogen bonds. The distance between
the N9 of purine and N1 of pyrimidine corresponding to a typical base pair is found to be
approximately 9 Å. The distance between the N atom of urea covalently bonded to the sugar
moiety and the paired base (N9 for purine and N1 for pyrimidine) were calculated and the
distributions are given in Figure 3.2.5a.
45
Figure 3.2.5: Probability distribution of the distances (in Å) between connecting nitrogen atoms
of nucleobase or urea with furanose sugar for (a) urea-nucleobase mismatched pairs and (b) base
pairs next to the mismatched base pair (CG:red and AT:black) present in all the urea
incorporated B-DNA duplexes.
The distributions involving urea-GUA/ADE pairs sample the distance close to 9 Å with minor
deviation for G1a. However, for both the urea-THY and urea-CYT pairs, two distinct states
centered at approximately 7.5 and 9.5 Å are sampled with no significant probability in the
intermediate distances. Careful analysis of the trajectories indicates that they exist in dynamic
46
equilibrium between two states corresponding to these distances. Time series plot of the
distances indicate that the systems alternate between these two states in the ns timescale.
Analysis of the structures reveal that the state corresponding to a distance close to 7.5 is when
there is direct hydrogen bond between the nucleobase and urea, whereas the other state
corresponds to the state where the two moieties interact via bridging water molecules (Figure
3.2.6).
Figure 3.2.6: Two modes of interactions between urea and pyrimidine bases. Depiction of direct
hydrogen bonds and water mediated hydrogen bonds of urea with its complementary bases,
thymine (a:T2c) and cytosine (b:C2b), during the course of the molecular dynamics simulations.
Such dynamics will lead to detection of the imino protons of this pair difficult/impossible.
Notably, previous NMR studies had difficulties in studying nucleic acid structures with urea-
47
nucleobase pairs possibly because of this effect. This phenomenon also is expected to alter the
solvation dynamics of the nucleic acid significantly [26, 28]. To understand this further, the
number of water molecules present around the urea:nucleobase pair were calculated. Table 3.2.2
shows the average numbers of water molecules around the urea-nucleobase pairs, and these
values indicate that more number of water molecules are present around the urea-pyrimidine
base pairs compared to urea-purine base pairs. This is because of the difference in mode of
interaction of urea lesion with purine and pyrimidine bases. As discussed above, urea lesion
forms direct hydrogen bonds with purine bases while for urea-pyrimidine base pairs, water
mediated hydrogen bonds are also observed in addition to direct hydrogen bonds and an
equilibrium between these two states (Figure 3.2.6). No such water mediated hydrogen bond
formations are found when the urea lesion forms hydrogen bonds with purine nucleobases. This
further indicates that water molecules play a significant role in stabilizing the urea-pyrimidine
base pairs in DNA double helix.
Table 3.2.2: Number of water molecules present around the mismatched base pair (urea and
complementary base) for all the urea incorporated B-DNA duplexes considered in the present
study.
Duplex
Hydration
Number
A2b
10.57 ± 0.04
G1a
11.72 ± 0.04
G1c
10.49 ± 0.02
T2c
13.43 ± 0.04
C2b
13.23 ± 0.05
48
Figure 3.2.7: Probability distributions of pseudorotation angles of furanose sugars of urea
nucleotides (Ur) and their respective complementary nucleotides (cU) in all the urea incorporated
B-DNA duplexes. The probabilities corresponding to Ur and cU are shown in blue and green
respectively, while the probability corresponding to pure DNA duplexes is shown in purple.
49
Local and global conformational properties of B-DNA duplexes after the incorporation of
the urea nucleotide:
Conformational properties of the DNA duplexes and changes in their conformation after the
incorporation of urea lesion were examined by calculating the pseudorotation angles and
characteristic backbone dihedral angles such as α (O3’-P-O5’-C5’), β (P-O5’-C5’-C4’), γ (O5’C5’-C4’-C3’), ε (C4’-C3’-O3’-P), and δ (C3’-O3’-P-O5’).
The probability distributions of pseudorotation angles indicate that the conformation of
the furanose sugars of urea nucleotide depends on its complementary base (Figure 3.2.7). The
deoxyribose sugars generally sample populations majorly in C2'-endo and minor populations in
O4'-endo regions [61, 62]. The furanose sugars corresponding to urea lesions opposite to guanine
and cytosine predominantly sample in C2'-endo regions whereas furanose sugars corresponding
to urea lesion complementary to adenine and thymine nucleobases sample conformations in C2'endo and C3'-endo regions. The changes in pseudorotation profiles of furanose sugars of urea
lesion are restricted to local changes and do not alter the global conformation of B-DNA
duplexes (Figure 3.2.8a and b).
Similar conformations have been observed in NMR studies for the furanose sugars
corresponding to the modified sites in B-DNA duplexes with urea or formamide lesions [26, 28].
Sampling of the population in O4’-endo region increases for the furanose sugars of nucleobases
that are opposite to urea lesion. To understand the conformation of the urea and complementary
nucleotides, glycosidic dihedral angles corresponding to sugar-base or sugar-urea bond were
calculated.
50
Figure 3.2.8: Probability distributions of pseudorotation angles of furanose sugars for the
(a) full DNA duplex, (b) full DNA duplex without urea-nucleobase pair, and (c, d) neighbour
base pairs in all the urea incorporated DNA duplexes. The pseudorotation angles corresponding
to the furanose sugars of typical B-DNA duplexes are shown in black while the urea
incorporated DNA duplexes are shown in red.
Probability distributions shown in Figure 3.2.9a indicate that all the urea lesions exhibit
anti conformation except for guanine in G1a conformation, in which urea exhibits both syn and
anti-conformations. As shown in Figure 3.2.9b, the probability distributions indicate that the
51
complementary purine bases sample conformations that are sampled by the bases in the regular
B-DNA duplex. However, the pyrimidine bases sample regions corresponding to regions
sampled by bases in regular B-form and A-form duplexes. This might be associated with the
change in pseudorotation profile of the furanose sugar that requires rearrangement in the
orientation of sugar-base glycosidic dihedral angle. The urea incorporation does not induce
considerable changes in the backbone conformation as revealed by the probability distributions
of the characteristic backbone dihedral angles corresponding to the urea nucleotide and its
neighboring nucleotides (Figure 3.2.10-3.2.11).
Figure 3.2.9: Probability distributions of sugar-base glycosidic angle (in degree) for urea
nucleotides (a) and respective complementary nucleobases (b) of all the urea incorporated BDNA duplexes.
52
This suggests that incorporation of urea lesion does not alter the backbone conformation
of the attached DNA strand significantly. The incorporation of the urea lesion preserves the Bform characteristics of the DNA duplexes, which is in agreement with previous studies [23].
Figure 3.2.10: Probability distributions of backbone dihedral angles (in degree) corresponding to
the urea nucleotides, its complementary nucleotide and their neighbour nucleotides in all the urea
incorporated DNA duplexes.
53
Figure 3.2.11: Probability distributions of phosphodiester bond angle (in degree) corresponding
to the urea nucleotides, its complementary base and their neighbour nucleotides in all the urea
incorporated DNA duplexes.
Thermodynamic stability of urea incorporated duplexes:
To understand the effect of incorporation of urea lesion on the thermodynamic stability of
DNA duplexes, binding free energies corresponding to duplex formation were calculated using
MM-GBSA method (Table 3.2.3).
54
Table 3.2.3: Absolute (∆G) and relative (∆∆G) binding free energy values calculated for the
duplex formation of B-DNA duplexes considered in the present study. The relative binding free
energy values are calculated with respect to their pure DNA duplex. All the energy values are in
kcal mol-1.
∆G
∆∆G
Pure DNA
-120.4
0.0
A2b
-119.0
1.4
Pure DNA
-127.1
0.0
G1a
-121.0
6.1
G1c
-115.4
11.7
Pure DNA
-123.3
0.0
T2c
-111.9
11.4
Pure DNA
-123.0
0.0
C2b
-114.2
8.8
Duplex
(a) Adenine
(b) Guanine
(c) Thymine
(d) Cytosine
The relative binding free energy values are shown with respect to their corresponding
pure DNA duplexes, and these values indicate that the urea incorporation marginally decreases
the thermodynamic stabilities of B-DNA duplexes. It is also seen that the extent of
destabilization depends on the size of the nucleobase base complementary to urea lesion.
55
Reduction of duplex stability is high when the urea lesion replaces purine bases compared to
pyrimidine bases. This is because the replacement of purine bases results in urea-pyrimidine base
pairs which are stabilized by two modes of interactions (direct and water mediated hydrogen
bonds) during the base pair formation. The stabilization due to bridging water molecules are not
accounted in the free energy calculation due to the methodological limitations. However, if urea
replaces pyrimidine bases, it results in a urea-purine base pair that is stabilized by direct
hydrogen bond interactions between urea and purine bases. This kind of mode of interaction
might be the factor responsible for the lower impact on the thermodynamic stabilities of DNA
duplexes when replacing pyrimidine bases compared to purine bases. This indicates that urea can
potentially replace the pyrimidine bases in purine-pyrimidine base pairs and mimic the
pyrimidine without significantly altering the duplex properties. Future studies can possibly
include DNA duplexes with homo AT and GC base pairs in which all the pyrimidine bases are
replaced by urea. This can provide further support for the pyrimidine-mimicking nature of urea.
56
Chapter 4
CONCLUSIONS
Atomistic molecular dynamics simulations were performed on a number of B-DNA
duplexes with the urea lesion complementary to all the four nucleobases (A, G, C and T). The
nature of hydrogen bonding between urea and all the four nucleobases were considered from
earlier quantum mechanical studies, and this bonding included WC, Hoogsteen and sugar edgedtype interactions. Several structural and energetic calculations were performed on the resultant
trajectories, and the results suggest that the urea incorporation preserves most of the global
characteristics of typical B-DNA duplexes despite small variations being seen around the
mismatched site for urea-pyrimidine base pairs. It was observed that within a DNA duplex, the
urea-nucleobase pairs with WC hydrogen bonding interactions were stable, whereas other
interacting conformations were either unstable or were transformed into WC conformations
during the course of the simulations. Urea lesions can form stable hydrogen bonds with
nucleobases and interact strongly with them which is evident from the hydrogen bond distances
and base pair interaction energy calculations. This is in good agreement with the previous results
obtained from NMR and quantum mechanical studies. The base pair interaction energies of ureapurine base pairs are comparable to the values of pyrimidine-purine base pairs that are present in
canonical B-DNA duplex. The interaction energy values corresponding to urea-pyrimidine base
pairs, however, were small compared to typical base pair values. Nevertheless, all the ureanucleobase pairs were quite stable and the urea lesions stayed inside the helix during the course
of the simulations. Urea lesions can also form strong stacking interactions with the neighboring
nucleobases of the same strand and in the complementary strand. The incorporated urea lesions
57
experience an environment similar to regular bases that are present in the same and partner
strands. These urea-nucleobase pairs do not alter the geometries of the neighboring base pairs.
The formation of hydrogen bonds directly between the urea lesion and the purine bases stabilizes
the base pairs, whereas water mediated hydrogen bonds were also observed in case of ureapyrimidine base pairs in addition to direct hydrogen bonds. These observations suggest that the
stability of mismatched base pairs depends on the size of the complementary base paired with the
urea lesions. Free energies corresponding to the duplex formation were further calculated to
understand the thermodynamic stability of DNA duplexes after the incorporation of urea lesion.
All these calculations support the fact that the urea lesion can potentially mimic nucleobases in
typical B-DNA duplexes without affecting the helical integrity significantly.
In vitro studies have shown that DNA polymerase stops at one nucleotide upstream of a
urea lesion [15]. The present study suggests that the mismatched urea-nucleobase pairs are quite
stable and can potentially replace base pairs in typical B-DNA duplexes. It is also observed that
there is no equilibrium between intrahelical and extrahelical conformations as observed for
abasic sites and bulges. There have been a few studies that attempt to understand ureanucleobase pairs, especially T and G, in the context of B-DNA duplex, and these show that ureathymine base pair is quite stable [23]. Due to the capability of forming strong hydrogen bonds
with all four nucleobases, DNA polymerase can incorporate any base complementary to urea
lesion unlike formamide lesion, which is specific to guanine. This leads to the existence of
several possibilities to mutate the DNA sequence. Another interesting observation is that urea
can interact with guanine base in multiple ways that are stable throughout the simulations. This
suggests that DNA polymerase can specifically incorporate guanine opposite to urea. This is not
a general observation reported in experiments, although formamide lesions have been shown to
58
be specific to guanine [29]. Decreased stacking interaction energies could be one reason why
DNA polymerase stops one nucleotide upstream of the urea lesion. Replacement of the existing
nucleobase with urea lesion results in the decrease of stacking interactions in the nucleobases
around the mismatched site, which might affect the activity of DNA polymerase.
59
Related Publications
1. Kasavajhala, K., Bikkina, S., Patil, I. and MacKerell, A.D. Jr. and Priyakumar, U.D.
(2015) Dispersion interactions between urea and nucleobases contribute to the
destabilization of RNA by urea in aqueous solution. J. Phys. Chem. B 119, 3755-3761.
2. Gorle, S., Patil I. and Priyakumar, U.D. Urea Mimics Nucleobases by Preserving the
Helical Integrity of B-DNA Duplexes via Hydrogen Bonding and Stacking Interactions.
(Submitted)
60
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