Abstract

Hairpin DNA structures that are formed due to overexpansion of CAG repeat lead to Huntington’s disorder and spinocerebellar ataxias. Nonetheless, DNA hairpin stem structure that generally embraces B-form with canonical base pairs is poorly understood in the context of periodic noncanonical A. . .A mismatch as found in CAG repeat overexpansion. Molecular dynamics simulations on DNA hairpin stems containing A. . .A mismatches in a CAG repeat overexpansion show that

.A dictates local Z-form irrespective of starting glycosy/ conformation, in sharp contrast to canonical DNA duplex. Transition from B-to-Z is due to the mechanistic effect that originates from its pronounced nonisostericity with flanking canonical base pairs facilitated by base extrusion, backbone and/or base flipping. Based on these structural insights we envisage that such an unusual DNA structure of the CAG hairpin stem may have a role in disease pathogenesis. As this is the first study that delineates the influence of a single A. . .A mismatch in reversing DNA helicity, it would further have an impact on understanding DNA mismatch repair.

Author Summary

Such DNA sequences tend to form unusual DNA structures comprising of base pairing schemes different from the canonical A. . .T/G. . .C base pairs. Influence of such unusual base pairing on the overall 3-dimensional structure of DNA and its impact on the pathogenesis of disorder is not well understood. CAG repeat overexpansion that leads to Huntington’s disorder and several spinocerebellar ataxias forms noncanonical A. . .A base pair in between canonical C. . .G and G. . .C base pairs. However, no detailed structural information is available on the influence of an A. . .A mismatch on a DNA structure under any sequence context. Here, we have shown for the first time that A. . .A base pairing in a CAG repeat provokes the formation of left-handed Z-DNA due to the pronounced structural dissimilarity of A. . .A base pair With G. . .C base pair, leading to periodic B-Z junction. Thus, these results suggest that formation of periodic B-Z junction may be one of the molecular bases for CAG

repeat instability.

Introduction

It is well known that formation of such unusual non-B-DNA structures during the overexpansion of trinucleotide microsatellites (tandem repeats of 1—3 nucleotide length) is responsible for at least 22 incurable trinucleotide repeat expansion disorders (TREDs) that are mainly neurological or neuromuscular in nature[1,2,3,4,5]. For instance, occurrence of hairpin structure due to the abnormal increase in the CTG repeat length in the untranslated region of DMPK gene causes myotonic dystrophy type-1[6,7]. Likewise, hairpin formation in CAG repeat expansion located in the protein-coding region leads to Huntington’s disorder & several spinocerebellar ataxias[7]. Direct evidence for the role of such hairpin structure in instigating replication-dependent instability has been demonstrated for the first time in human cells with 5’CTG.5’CAG microsatellite overexpanion[8]. Recently, it has been shown that CAG repeat overexpansion in DNA leads to toxicity by triggering cell death[9,10] and thus, warranting a detailed investigation on the hairpin structures formed under such abnormal expansion.

In fact, crystal structures of RNA duplex (hairpin stems) containing CUG[12] and CAG[13] repeats that form noncanonical U. . .U[12] & A. . .A[13] base-pairs offers useful information as the pathogenic CUG and CAG RNA hairpins have a role in misregu-lating the alternative splicing by MBNLl [14], leading to neurotoxicity. Though the isosequential DNA also intends to form hairpin structure[15], detailed structural insights about DNA duplex with CAG and CTG repeats that form A. . .A and T. . .T mismatches respectively are still inaccessible. With emerging evidence on ‘DNA toxicity’ of CAG repeat overexpansion[9,10] , such structural information would facilitate the understanding of underlying mechanisms behind repeat instability at DNA level which is yet another potential drug target. In this context, we aim here to investigate the structure and dynamics of DNA duplex containing CAG repeat using molecular dynamics (MD) simulation technique. Surprisingly, results of the MD simulations indicate that A. . .A mismatch in a CAG repeat overexpansion induces periodic B-Z junction irrespective of the starting conformation. Thus, we suggest that such an unusual DNA structure of GAG hairpin stem may affect the biological function and may be one of the factors responsible for ‘DNA toxicity’ [9,10].

Results

. .A23 pair amidst canonical base pairs (Fig. 1A) is investigated through 300ns MD simulation, prior to the investigation of CAG repeats With periodic Root mean square deviation (RMSD) calculated over 300ns simulation indicates the existence of three different ensembles (Fig. 1B): the first ensemble persists till ~16.5ns with RMSD centered around 2.8(0.7)A, the second one persists between 16.5-181ns with a RMSD of 4.7(0.7)A and the third one persists beyond ~181ns with the highest RMSD of 6.2(0.8)A. Intriguingly, a high RMSD of 4.5(0.6)A observed between 16.5-100ns is associated with a change in glycosyl conformation of mismatched A23 and A8 from the starting anti conformation to syn conformation.

1C). Thus, it is clear that A8. . .A23 mismatch disfavors anti. . .anti glycosyl conformation and causes distortion in the duplex.

1G) flanked by high (positive) twists at the neighboring G6C7 (32 (4°)) & A8G9 (31(6°)) steps (81 Fig). These, together with the conformational changes at A23. . .A8 mismatch reflect in the helicity of the duplex, which can be clearly seen from the superposition of average structures calculated over 1-100ps and 14.9-15ns (Fig. 1H). While the former is in B-form conformation, the latter shows a change in helicity leading to local Z-DNA formation. Occurrence of a low negative twist due to local Z-DNA formation in the midst of high twists at G6C7A8G9 stretch leads to local unwinding of the helix as can be seen Fig. 11. As A23. . .A8 mismatch site is located exactly in the middle of DNA (Fig. 1A), aforementioned distortions lead to Z-DNA sandwich, viz., a mini Z-DNA is embedded in a B-DNA. Essentially, similar features are observed in B-Z junction formed by L-deoxy guanine and L-deoxy cytosine (81 Fig).

During the first ~16.5ns, N1(A8). . .N6(A23) hydrogen bond persists, whereas, between 16.5-100ns, N1(A23). . .N6(A8) hydrogen bond is predominantly favored due to the slight movement of A23 towards the minor groove. Base extrusion at the mismatch site is also observed during 100ns simulation. Concomitant to above, major and minor groove widths also undergo changes. Unwinding of the helix leads to the expansion of minor groove width at the mismatch site to ~20.1(0.4)A flanked by comparatively narrower groove widths of 12.7A&14.9A on either side at the end of the 300ns simulation.

2D plots indicating backbone conformational preference for d(CAG)ZCAG(CAG)2.d(CTG)2CAG (CTG)2 duplex with A8. . .A23 in anti. . .anti starting glycosyl conformation during the last 10ns. (5&0 and (0&0 2D plots corresponding to the first strand is given in 2nd column along with the appropriate step marked in the 1St column. (5&0 and (0&0 2D plots corresponding to the second strand is given in 4th column along with the appropriate step marked in the 3ml column. 2D plots of (5&0 and (0&0 are marked in black & red respectively. Note that 5&0 are represented in X-axis and C&v are represented in Y-axis.

. .A23 mismatch site to accommodate the mismatch. In fact, an increase in Z-DNA stretch around the mismatch site is seen (Fig. 3B) during the 300ns simulation. One of the marked changes associated With Z-DNA conformational preference is A8 adopting high-anti/—syn (287 (17°)) glycosyl conformation beyond 36ns (Fig. 3C). Conformational changes at A8 beyond 36ns enforce syn glycosyl conformation for neighboring G9 (248 (26°) to 321(32°)) and G24 (248(25°) to 324 (15°)) (83A Fig). Other notable changes that happen during the early part of the simulation (~9ns) in seeding Z-DNA conformation are, the preference for syn glycosyl conformation by G21 (from 249(24°) to 296 (25°)) (hydrogen bonded With C10) and All (from 257(23°) to 302(32°)) (base paired With T20) that are located in the neighborhood of A8. . .A23 mismatch site (83A Fig). Irrespective of the above conformational changes, chi at A23 stays close to the initial +syn (83A Fig) conformation throughout the simulation. It is noteworthy that a total loss of hydrogen bonds at N1 (A8). . .N6 (A23) & N6(A8). . .N7(A23) that happenes due to base extrusion during 30-40ns facilitates B-Z transition (S3B-E Fig).

4) that is facilitated by the Z-DNA conformation. As a result, there is a total loss of N1(A8). . .N6(A23) hydrogen bond as well as N6(A8). . .N7(A23) hydrogen bond between the mismatched bases beyond 150ns (S3B Fig). It happens in such a way that ~133ns the hydrogen bond becomes longish, followed by A8 and A23 moving out-of-plane with each other. Subsequently, A8 stacks on top of A23 like an intercalator and stays till the end of the simulation (Fig. 4). During the aforementioned conformational changes, the canonical

1), formation of local Z-DNA conformation is propagated to the neighboring bases (from C7 to G12) of A8. . .A23 mismatch. This eventually reflects in at least 3 steps located in the middle of the dupleX taking up lower helical twists (S5B-D Fig). Essentially, this leads to unwinding of the double heliX (S5B-D Fig & S7 Fig and S4 Movie), a typical characteristic of B-Z junction (PDB ID: 1FV7). Such unwinding is accompanied by eXpansion in the major (maximum of 28 A) and minor (maximum of 20 A) groove widths. However, at the mismatch site, the minor groove width shrinks to 11.5 A during the lOOns simulation. It further shrinks to 8 A, followed by the stacked conformation of A8&A23. Thus, formation of a local Z-DNA conformation accompanied by unwinding of the helix is evident even With a single A. . .A mismatch irrespective of the starting conformation.

Periodic B-Z junction in (CAG)6.

(CAG)6 duplex

.A mismatch as in the real situation of Huntington’s disorder and several spinocerebellar ataXias, 300ns MD simulation has been carried out for d(CAG)6.d(CAG)6 sequence (Fig. 5A). As before, 2 starting models each With +syn. . .anti and anti. . .anti glycosyl conformations are considered for all the siX

.A pair with anti. . .anti starting conformation. A high RMSD of 8.2(0.5)A beyond 25ns (Fig. 5B) implicates that the initial model With A. . .A mismatches in anti. . .anti starting conformation undergoes signif1cant conformational rearrangement to accommodate the mismatches.

.A mismatch at the CA and GC steps leads to sugar-phosphate backbone flipping causing helicity reversal that results in the formation of periodic B-Z junction (Fig. 51). Formation of such B-Z junction also reflects in the solvation as both water and ion populate more in the minor groove than the major groove (310 Fig).

. .N1(A23) due to the movement of A23 towards the minor groove and stays till ~162ns. Just in 200ps (between 162—162.2ns), base flipping occurs accompanied by syn glycosyl conformation for A23&A14 (Fig. 6(top), 811 Fig and SS Movie). Similarly, at A26. . .Au mismatch site, base extrusion happens ~205ns resulting in a total loss of

6 (bottom), 815 Fig). These indicate the periodic occurrence of B-Z junction in d(CAG)6.d(CAG)6. Above conformational rearrangements result in a high RMSD of ~8A at the end of the simulation (816 Fig). Further, similar to above (810 Fig), B-Z junction results in minor groove of the dupleX occupied with more water and ion molecules compared to the major groove (817 Fig), a characteristic of the Z-DNA.

.A mismatch favors (i)syn. . .high-anti/(—)syn conformation over anti. . .anti and +syn. . .anti glycosyl conformation and invokes B-Z junction. Formation of B-Z junction takes place either through base flipping or through backbone flipping Without affecting the canonical G. . .C and C. . .G hydrogen bonding pattern (818 Fig).

Canonical (CTG)6.

(CAG)6 duplex retains B-form

7A) indicates that the molecule undergoes minimal conformational rearrangement from the starting B-form geometry (Fig. 7B). Strikingly, the overall structure doesn’t show any tendency to adopt Z-form, as can be Visualized from Fig. 7C. Instead, it retains the compact B-form geometry.

Discussion

.A mismatch in a DNA duplex is not yet well defined at the atomistic level. The only structure that has been reported so far With A. . .A mismatch in a DNA is the complex of a DNA duplex and Muts, an E. coli mismatch repair protein, With a significant bending at the mismatch site (PDB ID: 2WTU). NMR and thermodynamic studies of A. . .A mismatch containing DNA duplex offer controversial results. While some of them suggest that A. . .A mismatch destabilizes[18,19,20,21] the DNA duplex significantly, the others do not[22]. Physicochemical studies indicate that A. . .A mismatch in a GAC repeat adopt several distinct conformations in solution including Z-DNA[23,24]. In fact, it has been suggested that A. . .A mismatch in GAC repeat promotes Z-DNA formation [23].

.A mismatch is very important in the context of Huntington’s disorder and several spinocerebellar ataxias due to the formation of hairpin structures consisting of noncanonical A. . .A base-pairs. MD simulations carried out in this context reveal a very exquisite observation that A. . .A mismatch in a CAG repeat induces change in the helicity from right-handed B-DNA to left-handed Z-DNA. Even a single A. . .A mismatch tends to form a local Z-DNA structure leading to Z-DNA sandWich (Figs. 1,3). When the A. . .A mismatches occur in a regular interval, it leads to local left-handed Z-DNA formation at the mismatch site followed by a right-handed DNA at the canonical WC pair site leading to periodic B-Z junctions (Figs. 5,6). Formation of Z-DNA structure is evident from the preference for (i)syn. . .high-anti/(—)syn glycosyl conformation by A. . .A mismatch and backbone conformational angles (agony) favoring (g',g+,g+,t), (g',g',g+,t) and (g',g',g',g+) at & around the mismatch site. Additionally, G’s prefer syn conformation. This results in a low helical tWist at the CA and AG steps in the midst of high tWist at the GC step, a characteristic of B-Z junction (PDB ID 1FV7).

Mechanism of formation of 8-2 junction

.A mismatch induces Z-DNA conformation through ‘zipper mechanism’ [25] assisted by base extrusion, base and/or backbone flipping (Figs. 1,6 and SZ,S3&821 Figs). While the sugar-phosphate backbone flipping is prominent in anti. . .anti glycosyl conformation, base extrusion and sugar-phosphate & base flipping are favored by +syn. . .anti conformation to transit from B-to-Z form DNA. Yet another interesting fact is that the above-mentioned Z-DNA formation is a noninstanta-neous event, rather it propagates in a stepwise manner (Figs. 51, 6 (Bottom) and S7 Fig). Though the noncanonical A. . .A mismatch impels Z-DNA conformation, the canonical base pairs have the prevalence for B-form geometry resulting in B-Z junction. Formation of such B-Z junction can be readily visualized by unWinding of the double helix irrespective of the starting glycosyl conformation (822 Fig).

Base flipping mechanism

A. . .A mismatch adopts 2 different ‘base flipping’ pathways to undergo transition from

. .anti to syn. . .-syn (Fig. 6) accompanied by sugar phosphate rearrangements. One mode of transition is +syn moving to syn through cis conformation (Via counterclockwise rotation around glycosidic bond), While the other is Via trans conformation (through clockwise rotation around the glycosidic bond). In general, DNA With +syn. . .anti conformation takes longer time to undergo the B-Z transition, compared to anti. . .anti conformation.

Base pair nonisomorphism is the key factor for inducing Z-DNA conformation by A. .

.A mismatch

.A mismatch can be attributed to the higher degree of nonisomorphism between A. . .A mismatch and the canonical base pairs. This can be visualized from the larger value of residual twist and radial difference [17,26] , the measures of base pair nonisomorphism (S23 Fig). In fact, both residual twist (16°) and radial difference (1.6A) are quite prominent for A. . .A mismatch with anti. . .anti glycosyl conformation, but, only residual twist (16°) is significant and the radial difference is negligible (0.2A) in the case of +syn. . .anti glycosyl conformation. This may be the reason for the reluctance of A. . .A mismatch to retain anti. . .anti conformation and the transition to syn. . .-syn being quite fast compared to +syn. . .anti starting conformation.

In fact, several mechanisms have been proposed for B-to-Z transition[27] and a recent adaptively biased and steered MD study demonstrates the coexistence of Zipper and stretch-collapse mechanisms engaged in transition[28]. However, the mechanistic effect that arises from the intrinsic extreme nonisosterecity of A. . .A mismatch with the canonical base pairs immediately dictates B-to-Z transition without the influence of any external factors. As the A. . .A mismatch is single hydrogen bonded, it exhibits enormous flexibility for base extrusion and flipping, facilitating the formation of Z-DNA through Zipper mechanism. Interestingly, such a conformational change is not seen in the crystal structure of RNA duplex with A. . .A mismatch[13]. Thus, it is clear that the effect of A. . .A nonisomorph-ism is pronounced in the DNA and not in the RNA.

.A mismatches are prone to adopt parallel homodu-plex. Such preponderance for parallel duplex by these sequences may be due to left-handed Z-DNA provoking nature of A. . .A mismatch, which is a high-energy conformation. Hitherto, this aspect is not realized as there is no DNA duplex structure with A. . .A mismatch available with any sequence context. Earlier low-resolution 1D NMR studies on DNA duplexes comprising of A. . .A mismatch[18,19,20,21,22] offer only minimal information with some of them indicating notable destabilization induced at A. . .A mismatch site[18,19,20,21]. Strikingly, it has been shown by circular dichroism study that CAG repeat spectra resembles GA homoduplex but not CCG and CTG[32]. Propensity of A. . .A mismatch containing DNA to adopt a parallel DNA duplex is also reported[21]. However, the possibility of CAG repeat expansion to favor parallel duplex can be ruled out as it forms hairpin structure[7,8] , which eventually leads to an-tiparallel orientation for the two strands of the DNA hairpin stem. Thus, DNA hairpin stems containing CAG repeat may adopt local Z-DNA conformation at A. . .A mismatch site leading to ‘B-Z junction’ as revealed by the current investigation. Our result gains support from earlier surface probing using anti-DNA antibody that demonstrated the presence of Z-DNA structure in CAG & CTG repeat expansions [33]. It can also be recalled that formation of hairpin structure with such Z-DNA stem has been observed earlier in a different sequence context [34,35,36]. Thus, we envisage that such noncanonical ‘B-Z junction’ in CAG repeat expansion may be one of the factors responsible for the newly emerging mechanism of ‘DNA toxicity’ observed in CAG repeat expansion[37].

.A mismatch in a DNA duplex with CAG repeat is an inducer of local Z-form conformation through ‘zipper mechanism’ that stems from backbone flipping and base pair extrusion & flipping leading to B-Z junction. Such B-Z junction instilled by A. . .A mismatch results from the mechanistic effect intrinsic to the nonisoterecity of A. . .A mismatch with the flanking canonical base pairs. With emergence of evidence on ‘DNA toxicity’ of GAG overexpansion and its role in triggering cell death [9,10], one can envision that occurrence of B-Z junction is the molecular basis for Huntington’s disorder and several spinocerebellar ataxias. This further leads to the speculation that B-Z junction binding protein may have a role in the diseased states. Reported results would further be useful in understanding DNA repair mechanisms involving A. . .A mismatch, thus adding a new dimension to the role of A. . .A nonisosterecity on DNA structure.

Methods

Modeling of DNA duplex with A. . .A mismatch

. .N1(A) hydrogen bond. For the generation of model With periodic A. . .A mismatches (18mer, Fig. 3A), ‘T’s in the (CTG.CAG)6 dupleX are replaced manually With A’s as mentioned above. To establish base-sugar connectivity and to restraint the sugar-phosphate backbone conformation, the models are refined using X-PLOR [39] by constrained-restrained molecular geometry optimization and van der Waals energy minimization. The second conformation for the A. . .A mismatch, viz., N6(A). . .N1(A) hydrogen bond With +syn. . .anti glycosyl conformation is generated using X-PLOR by applying appropriate restraints. Subsequently, the models are subjected to a total of 1.5us molecular dynamics simulations (MD) using Sander module of AMBER 12 package [40].

Molecular dynamics simulation protocol

.A mismatches and the 3DNA generated canonical (CTG.CAG)6 duplex are solvated With TIP3P water molecules and net-neutralized With Na+ counter ions. Following the protocols described in our earlier papers [17,41,42] , equilibration and production runs are pursued for 300ns for the sequences given in Table 1. Simulations are performed under isobaric and isothermal conditions With SHAKE (tolerance = 0.0005 A) on the hydrogens [43], a 2fs integration time and a cutoff distance of 9 A for Lennard-Jones interaction. FF99SB forcefleld is used and the simulation is carried out at neutral pH. Trajectories are analyzed using Ptraj module of AMBER 12.0. Helical parameters and conformation angles are extracted from the output of 3DNA using in-house programs. Due to the presence of non-canonical base pairs, helical tWist angles are calculated With respect to Cl’. . .Cl’ vector [17,41,42]. Pymol is used for Visualization and MATLAB software (The MathWorks Inc., Na-tick, Massachusetts, United States) is used for plotting the graphs.

Supporting Information

backbone flipping at the mismatch site leading to the formation of B-Z junction. (MOV)

Base flipping leading to the formation of B-Z junction at A14. . .A23 mismatch site in d(CAG)6.d(CAG)6 DNA duplex with +syn. . .anti starting conformation for the mismatch (Fig. 5A). Note that one of the A’s moves towards minor groove and undergoes flipping

Base flipping leading to the formation of B-Z junction at A11. . .A26 mismatch site in d(CAG)6.d(CAG)6 DNA duplex with +syn. . .anti starting conformation for the mismatch (Fig. 5A). Note that prior to flipping, both the A’s are moving apart that results in total loss of hydrogen bond and subsequently, one of the A’s flips by rotating in clockwise direction. (MOV)

. .A23 mismatch and formation of Z—DNA sandwich. Comparison of B-Z junction formed by A8. . .A23 mismatch (T op-Left & T op-middle, current study) and by L-deoxy guanine and L-deoxy cytosine (T op-Right, PDB ID: 1FV7, Lowest energy structure). (Bottom) Sequence vs helical twist angle of the central 11-mer (Fig. 1A) corresponding to the average structure calculated over 99.9-100ns (Bottom-Left) and 299.9-300ns (Bottom-middle). Note the low helical twist at the mismatch site. Similar trend is also seen in B-Z junction induced by L-deoxy guanine and L-deoxy cytosine (Bottom-Right, PDB ID: 1FV7, Lowest energy structure) lead-A23. . .A8 (Left) that arises due to the stacked conformation of A23&A8, While the canonical

. .G24 and G9. . .C22 retain their hydrogen bonds. (CE) Different hydrogen bonding patterns observed for A8. . .A23 during the simulation. Note the total loss of hydrogen bond in (E) that happens between 30-40ns.

. .A23 mismatch on the sugar-phosphate backbone conformation. 3D plots showing the relationship between 8 & C and CL & y With respect to time in the case of d (CAG)2C§G(CAG)2.d(CTG)2C§G(CTG)2 duplex With +syn. . .anti glycosyl starting conformation. The corresponding step is indicated on top of the 3D plot.

. .A23 mismatch in +syn. . .anti starting glycosyl conformation. Sequence vs helical twist angle (central 11-mer) and the corresponding average structure (cartoon representation) calculated over (A) 0.09—0.1ns (B) 99.9-100ns (C) 149.9-150ns and (D) 299.9-300ns. Note that the low helical twists at and around the mismatch site are sandwiched between high helical tWists (sequence vs twist profiles given in AD). A8. . .A23 mismatch is colored pink and 04’ atoms of the sugars are colored orange in the cartoon representation of the average structures. Dotted lines indicate the helical tWist angle corresponding to ideal B-form. Note the unWinding of the double heliX around the mismatch site.

. .A23 in +syn. . .anti starting glycosyl conformation during the last 10ns. (8&0 and (0c&y) 2D plots corresponding to the first strand is given in 2nd and 3rd columns respectively along With the appropriate step marked in the 1st column. (8&0 and (0&7) 2D plots corresponding to the second strand is given in 5th and 6th columns respectively along With the appropriate step marked in the 4th column. (TIF)

.A mismatch in anti. . .anti starting glycosyl conformation. Sequence vs helical twist angle calculated for the average structure over last 100ps showing a high twist at GC step and a 10W twist at CA and AG steps.

. .anti starting glycosyl conformation for A. . .A mismatch. (8&0 and (0&7) contour density plot corresponding to (AD) CA, (EH) AG & (IL) GC steps. Note that the first two columns belong to residues from C1 to G18 of the duplex, While the third and fourth belong to the complementary residues (C19 to G36) of the duplex. While the first and third columns indicate the relationship between 8 &C (e in X-axis and C in Y-axis), the second and fourth columns illustrate the relationship between oc & 7 (es in X-axis and y in Y-axis). Scaling used for contour density plot is shown in the 4th row. Note the strong preponderance for Z-form geometry by CA and GC steps.

. .A14 mismatch site. The corresponding simulation time scale is mentioned below the mismatch. (TIF)

. .anti starting glycosyl conformation for A. . .A mismatches. (Top) Histogram of twist angles calculated over 291-300ns. (Bottom) Sequence vs. twist angle corresponding to the average structure calculated over last 100ps. Note the lOW twist at the CA & AG steps and high twist at the GC step. Though CA step takes Wide range of helical twist (between -20° to +50°), it has preference for lOW twist in the range of -20 to +20 (~70%).

. .anti (black) and anti. . .anti (red) starting glycosyl conformation for A. . .A mismatch. Note that While the latter attains the RMSD of ~8 A very early in the simulation, the former attains the RMSD of ~8 A only ~200ns as indicated by solid arrows. Dotted double-headed arrows indicate two ensembles of structures in the case of +syn. . .anti starting glycosyl conformation: one With RMSD of ~5 A during 200ns and other With RMSD of ~8 A beyond 200ns.

Note that unlike in 888615 (Bottom) Figs, the helical twist stays close to 30° indicative of geometry close to B-DNA. is shown in the 4th row. (TIF)

.A mismatch site (colored red) leading to the formation of Z—DNA observed in d(CAG)6.d(CAG)6 duplex with A. . .A mismatch in anti. . .anti starting glycosyl conformation. Note that only the pentamer sequence is shown for clarity.

.A mismatch. Cartoon representation of central hex-amer corresponding to d(C7X8G9C10X11G12).d(C25A26G27C28A29G30), wherein X = T for canonical duplex (Top) and X = A for non-canonical duplex (Middle and Bottom). A. . .A mismatch With anti. . .anti and syn. . .anti glycosyl starting conformations are shown in the middle and bottom respectively. Note the smooth right-handedness in canonical duplex, Whereas, the A. . .A mismatch induced B-Z junction leads to opening of the double helix.

.C pair with noncanonical A. . .A mismatch showing the extent of base pair nonisomorphism. Residual twist and radial difference, the quantitative measures of base triplet nonisomorphism, are quite high between G. . .C and A. . .A (~16 is ~1.6A), When the latter is in anti. . .anti glycosyl conformation (Top). When A. . .A is in syn. . .anti glycosyl conformation only the residual twist is quite high and the radial difference is negligible (~16 is ~0.2A).

Acknowledgments

The authors thank High Performance Computing facility of IITH, Center for Development of Advance of Computing (Government of India), Ministry of Defence (Government of India) and Inter University Accelerator Center (Government of India).

Author Contributions

Performed the experiments: TR NKh NKo. Analyzed the data: TR NKh NKo. Contributed reagents/materials/ analysis tools: TR. Wrote the paper: TR.