DFT study of B-like conformations of deoxydinucleoside monophosphates containing Gua and/or Cyt and their complexes with Na+ cation

B-like minimum energy conformations of deoxydinucleoside monophosphate anions (dDMPs) containing Gua and/or Cyt and their Na+ complexes have been studied by the DFT PW91PW91/DZVP method. The optimized geometry of the dDMPs is in close agreement with experimental observations and the obtained minimum energy conformations are consistent with purine-purine, purine-pyrimidine, and pyrimidine-purine arrangements in crystals of B-DNA duplexes. All the studied systems are characterized by pyramidalization of the amino groups, which participate in the formation of unusual hydrogen bond between the carbonyl oxygen of the second base in the dGpdC, dCpdG dDMPs, and their Na+ complexes. In all the obtained structures the bases assume a nearly parallel disposition to each other and this effect is independent on the degree of their spatial superposition. From this it is concluded that the parallel disposition of the bases in the B-like single-stranded conformations is dictated by the sugar-phosphate backbone. Correspondingly, the base-base interactions attain a secondary role in the formation of these spatial structures. The formation of a weak C6-H6...O5' hydrogen bond between cytosine and the phosphate oxygen is reported, in agreement with experimental observations.     

The conformations of locked nucleic acids (LNA)

We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2'-O, 4'-C-methylene bridge. We have investigated a modified 9-mer single stranded oligonucleotide as well as three 9- and 10-mer modified oligonucleotides hybridized to unmodified DNA. The single-stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson-Crick base-pairing with all the nucleotides in anti-conformation. By use of selective DQF-COSY spectra we determined the ratio between the N-type (C3'-endo) and S-type (C2'-endo) sugar conformations of the nucleotides. In contrast to the corresponding single-stranded DNA (ssDNA), we found that the sugar conformations of the single-stranded LNA oligonucleotide (ssLNA) cannot be described by a major S-type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N-type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N-type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S-type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N-type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases.     

Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulations

Locked nucleic acid (LNA) is a chemically modified nucleic acid with its sugar ring locked in an RNA-like (C3′-endo) conformation. LNAs show extraordinary thermal stabilities when hybridized with DNA, RNA or LNA itself. We performed molecular dynamics simulations on five isosequential duplexes (LNA–DNA, LNA–LNA, LNA–RNA, RNA–DNA and RNA–RNA) in order to characterize their structure, dynamics and hydration. Structurally, the LNA–DNA and LNA–RNA duplexes are found to be similar to regular RNA–DNA and RNA–RNA duplexes, whereas the LNA–LNA duplex is found to have its helix partly unwound and does not resemble RNA–RNA duplex in a number of properties. Duplexes with an LNA strand have on average longer interstrand phosphate distances compared to RNA–DNA and RNA–RNA duplexes. Furthermore, intrastrand phosphate distances in LNA strands are found to be shorter than in DNA and slightly shorter than in RNA. In case of induced sugar puckering, LNA is found to tune the sugar puckers in partner DNA strand toward C3′-endo conformations more efficiently than RNA. The LNA–LNA duplex has lesser backbone flexibility compared to the RNA–RNA duplex. Finally, LNA is less hydrated compared to DNA or RNA but is found to have a well-organized water structure.     

Conformational features of benzo‐homologated yDNA duplexes by molecular dynamics simulation

yDNA is a base‐modified nucleic acid duplex containing size‐expanded nucleobases. Base‐modified nucleic acids could expand the genetic alphabet and thereby enhance the functional potential of DNA. Unrestrained 100 ns MD simulations were performed in explicit solvent on the yDNA NMR sequence [5′(yA T yA yA T yA T T yA T)₂] and two modeled yDNA duplexes, [5′(yC yC G yC yC G G yC G G)₂] and [(yT5′ G yT A yC yG C yA yG T3′)?(yA5′ C T C yG C G yT A yC A3′)]. The force field parameters for the yDNA bases were derived in consistent with the well‐established AMBER force field. Our results show that DNA backbone can withstand the stretched size of the bases retaining the Watson‐Crick base pairing in the duplexes. The duplexes retained their double helical structure throughout the simulations accommodating the strain due to expanded bases in the backbone torsion angles, sugar pucker and helical parameters. The effect of the benzo‐expansion is clearly reflected in the extended C1′‐C1′ distances and enlarged groove widths. The size expanded base modification leads to reduction in base pair twist resulting in larger overlapping area between the stacked bases, enhancing inter and intra strand stacking interactions in yDNA in comparison with BDNA. This geometry could favour enhanced interactions with the groove binders and DNA binding proteins., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 55–64, 2016     

Biologically important conformational features of DNA as interpreted by quantum mechanics and molecular mechanics computations of its simple fragments

Deciphering the mechanism of functioning of DNA as the carrier of genetic information requires identifying inherent factors determining its structure and function. Following this path, our previous DFT studies attributed the origin of unique conformational characteristics of right-handed Watson-Crick duplexes (WCDs) to the conformational profile of deoxydinucleoside monophosphates (dDMPs) serving as the minimal repeating units of DNA strand. According to those findings, the directionality of the sugar-phosphate chain and the characteristic ranges of dihedral angles of energy minima combined with the geometric differences between purines and pyrimidines determine the dependence on base sequence of the three-dimensional (3D) structure of WCDs. This work extends our computational study to complementary deoxydinucleotide-monophosphates (cdDMPs) of non-standard conformation, including those of Z-family, Hoogsteen duplexes, parallel-stranded structures, and duplexes with mispaired bases. For most of these systems, except Z-conformation, computations closely reproduce experimental data within the tolerance of characteristic limits of dihedral parameters for each conformation family. Computation of cdDMPs with Z-conformation reveals that their experimental structures do not correspond to the internal energy minimum. This finding establishes the leading role of external factors in formation of the Z-conformation. Energy minima of cdDMPs of non-Watson-Crick duplexes demonstrate different sequence-dependence features than those known for WCDs. The obtained results provide evidence that the biologically important regularities of 3D structure distinguish WCDs from duplexes having non-Watson-Crick nucleotide pairing.     

Interaction of methyl green with an oligonucleotide in intramolecular duplex and triplex conformations

Interaction of methyl green with the oligonucleotide 5′-dGGAAAAGG-[T₄]-GGAAAAGG-[T₄]-CCTTTTCC (where [T₄] is a nucleotide sequence of four thymines) in hairpin duplex and in intramolecular triplex structures has been studied by circular dichroism. We found that methyl green binding to the duplex form shows a complex pattern, exhibiting an exciton contribution when the number of bound molecules increases. Differences between this pattern and previously published results on other DNAs reveals the presence of different types of complexes. In contrast to previous findings with the triple helix poly(dA).2poly(dT) we show that the methyl green is not totally excluded from this triplex structure made of Pur:Pur:Pyr triplets. Received: 15 July 1997 / Accepted: 22 September 1997     

Mode‐coupling Smoluchowski dynamics of a double‐stranded DNA oligomer

The local dynamics of a double‐stranded DNA d(TpCpGpCpG)₂ is obtained to second order in the mode‐coupling expansion of the Smoluchowski diffusion theory. The time correlation functions of bond variables are derived and the ¹³C‐nmr spin–lattice relaxation times T₁ of different ¹³C along the chains are calculated and compared to experimental data from the literature at three frequencies. The DNA is considered as a fluctuating three‐dimensional structure undergoing rotational diffusion. The fluctuations are evaluated using molecular dynamics simulations, with the ensemble averages approximated by time averages along a trajectory of length 1 ns. Any technique for sampling the configurational space can be used as an alternative. For a fluctuating three‐dimensional (3D) structure using the three first‐order vector modes of lower rates, higher order basis sets of second‐rank tensor are built to give the required mode coupling dynamics. Second‐ and even first‐order theories are found to be in close agreement with the experimental results, especially at high frequency, where the differences in T₁ for ¹³C in the base pairs, sugar, and backbone are well described. These atomistic calculations are of general application for studying, on a molecular basis, the local dynamics of fluctuating 3D structures such as double‐helix DNA fragments, proteins, and protein–DNA complexes. © 1999 John Wiley & Sons, Inc. Biopoly 50: 613–629, 1999     

DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

The maintenance methyltransferase M.EcoKI recognizes the bipartite DNA sequence 5'-AACNNNNNNGTGC-3', where N is any nucleotide. M.EcoKI preferentially methylates a sequence already containing a methylated adenine at or complementary to the underlined bases in the sequence. We find that the introduction of a single-stranded gap in the middle of the non-specific spacer, of up to 4 nt in length, does not reduce the binding affinity of M.EcoKI despite the removal of non-sequence-specific contacts between the protein and the DNA phosphate backbone. Surprisingly, binding affinity is enhanced in a manner predicted by simple polymer models of DNA flexibility. However, the activity of the enzyme declines to zero once the single-stranded region reaches 4 nt in length. This indicates that the recognition of methylation of the DNA is communicated between the two methylation targets not only through the protein structure but also through the DNA structure. Furthermore, methylation recognition requires base flipping in which the bases targeted for methylation are swung out of the DNA helix into the enzyme. By using 2-aminopurine fluorescence as the base flipping probe we find that, although flipping occurs for the intact duplex, no flipping is observed upon introduction of a gap. Our data and polymer model indicate that M.EcoKI bends the non-specific spacer and that the energy stored in a double-stranded bend is utilized to force or flip out the bases. This energy is not stored in gapped duplexes. In this way, M.EcoKI can determine the methylation status of two adenine bases separated by a considerable distance in double-stranded DNA and select the required enzymatic response.     

In search of a Hoogsteen base paired DNA duplex in aqueous solution

When the oligodeoxynucleotides d(A)6 and d(T)6 are mixed together in a 1:1 ratio (in 100 mM NaCl), the NH signals in the NMR spectrum gave a typical signature of Watson-Crick paired (WC) and Hoogsteen paired (H) AT base pairs. The observation indicates two schemes: Scheme I, WC and H duplexes in slow equilibrium, i.e., WC in equilibrium with H, Scheme II, the WC helix formed is unstable and that it disproportionates into a triple helix (TR) and free d(A)6. We show that (i) addition of extra d(A)6 does not change the helix composition, (ii) addition of a minor-groove specific drug Dst2 (a distamycin analogue) results in an exclusive WC helix-drug duplex, while it does not destabilize triple helix in a 1:2 mixture. In addition we have compared the melting profile, 31P NMR spectra, 1H NMR spectra and the salt dependence of the 1:1 mixture and that of a pure triple helix. All the data from the above experiments overwhelmingly favor Scheme I. However Scheme II cannot be categorically excluded. Based on 1D/2D NMR studies, we have characterized the structural properties of the Hoogsteen double helix in terms of nucleotide conformations. In addition, we computationally demonstrate that the relative stability of the WC over the H duplexes increases with increasing chain length.     

Unique properties of purine/pyrimidine asymmetric PNA.DNA duplexes: differential stabilization of PNA.DNA duplexes by purines in the PNA strand

PNA.DNA duplexes are significantly stabilized by purine nucleobases in the PNA strand. To elucidate and understand the effect of switching the backbone in a nucleic acid duplex, we now report a thermodynamics study along with a solution conformations study of two purine/pyrimidine strand asymmetric duplexes and a strand symmetrical control by comparing the behavior of all four possible PNA/DNA combinations. In essence, we are comparing an identical basepair stack connected by either an aminoethyl glycine PNA or a deoxyribose DNA backbone. We show that the PNA.DNA duplexes containing purine-rich PNA strands are stabilized with regard to the thermal melting temperature and free energy as well as enthalpy (and concomitantly relatively less entropically disfavored). Based on our data, we find it unlikely that differences in counterion binding (identical ionic-strength dependence was observed), hydration (identical and insignificant water release was observed), or single-strand conformation can be responsible for the difference in duplex stability. The only consistent difference observed between the purine-rich PNA versus the pyrimidine-rich PNA in isosequential PNA.DNA duplexes is the significant increase in both binding enthalpy and entropy for the PNA.DNA duplexes containing pyrimidine-rich PNA in organic solvent, which would indicate that these duplexes are relatively enthalpically disfavored in water. Although our results so far do not allow us to identify the origin of the different stabilities of homopurine/homopyrimidine PNA.DNA duplexes, the evidence does point to a significant structural component, which involves enthalpic contributions both within the duplex structure and also from bound water molecules.     

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.     

Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion

Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, noncanonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype–phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction‐dependent destabilization of the underlying double helix, and a self‐structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self‐structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size‐dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 1–12, 2014.     

Mechanics of DNA flexibility visualized by selective 2'-amine acylation at nucleotide bulges

We used selective acylation of 2'-amine-substituted nucleotides to visualize local backbone conformations that occur preferentially at bulged sites in DNA duplexes. 2'-Amine acylation reports local nucleotide flexibility because unconstrained 2'-amino nucleotides more readily reach a reactive conformation in which the amide-forming transition state is stabilized by interactions between the amine nucleophile and the adjacent 3'-phosphodiester group. Bulged 2'-amine-substituted cytidine nucleotides react approximately 20-fold more rapidly than nucleotides constrained by base-pairing at 35 degrees C. In contrast, base-paired 2'-amine-substituted nucleotides flanked by a 5' or 3' bulge react two- or six-fold more rapidly, respectively, than the perfectly paired duplex. The relative lack of 2'-amine reactivity for nucleotides adjacent to a DNA bulge emphasizes, first, that structural perturbations do not extend significantly into the flanking duplex structure. Second, the exquisite sensitivity towards very local perturbations in nucleic acid structure suggests that 2'-amine acylation can be used to chemically interrogate deletion mutations in DNA. Finally, these data support the mechanical interpretation that the reactive ribose conformation for 2'-amine acylation requires that the base lies out of the helix and in the major groove, a mechanistic insight useful for designing 2'-amine-based sensors.     

NMR studies of abasic sites in DNA duplexes: deoxyadenosine stacks into the helix opposite acyclic lesions

Proton and phosphorus NMR studies are reported for two complementary nonanucleotide duplexes containing acyclic abasic sites. The first duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-P-C-A-T-G), contains an acyclic propanyl moiety, P, located opposite a deoxyadenosine at the center of the helix (designated APP 9-mer duplex). The second duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-E-C-A-T-G), contains a similarly located acyclic ethanyl moiety, E (designated APE 9-mer duplex). The ethanyl moiety is one carbon shorter than the natural carbon-phosphodiester backbone of a single nucleotide unit of DNA. The majority of the exchangeable and nonexchangeable base and sugar protons in both the APP 9-mer and APE 9-mer duplexes, including those at the abasic site, have been assigned by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H2O and D2O solution between -5 and 5 degrees C. These spectroscopic observations establish that A5 inserts into the helix opposite the abasic site (P14 and E14) and stacks between the flanking G4.C15 and G6.C13 Watson-Crick base pairs in both the APP 9-mer and APE 9-mer duplexes. The helix is right-handed at and adjacent to the abasic site, and all glycosidic torsion angles are anti in both 9-mer duplexes. Proton NMR parameters for the APP 9-mer and APE 9-mer duplexes are similar to those reported previously for the APF 9-mer duplex (F = furan) in which a cyclic analogue of deoxyribose was embedded in an otherwise identical DNA sequence [Kalnik, M. W., Chang, C. N., Grollman, A. P., & Patel, D. J. (1988) Biochemistry 27, 924-931]. These proton NMR experiments demonstrate that the structures at abasic sites are very similar whether the five-membered ring is open or closed or whether the phosphodiester backbone is shortened by one carbon atom. Phosphorus spectra of the APP 9-mer and APE 9-mer duplexes (5 degrees C) indicate that the backbone conformation is similarly perturbed at three phosphodiester backbone torsion angles. These same torsion angles are also distorted in the APF 9-mer but assume a different conformation than those in the APP 9-mer and APE 9-mer duplexes.     

Stabilization factors affecting duplex formation of peptide nucleic acid with DNA.

Peptide nucleic acid (PNA) is an oligonucleotide analogue in which the sugar-phosphate backbone is replaced by an N-(2-aminoethyl)glycine unit to which the nucleobases are attached. We investigated the thermodynamic behavior of PNA/DNA hybrid duplexes with identical nearest neighbors but with different sequences and chain lengths (5, 6, 7, 8, 10, 12, and 16 mers) to reveal whether the nearest-neighbor model is valid for the PNA/DNA duplex stability. CD spectra of 6, 7, and 8 mer PNA/DNA duplexes showed similar signal, while 10, 12, and 16 mer duplexes did not. The average difference in Delta G degrees (37) for short PNA/DNA duplexes with identical nearest-neighbor pairs was only 3.5%, whereas that of longer duplexes (10, 12, and 16 mers) was 16.4%. Therefore, the nearest-neighbor model seems to be useful at least for the short PNA/DNA duplexes. Thermodynamics of PNA/DNA duplexes containing 1--3 bulge residues were also studied. While the stability of the 12 mer DNA/DNA duplex decreased as the number of bulge bases increases, the number of bulge bases in PNA/DNA unchanged the duplex stability. Thus, the influence of bulge insertion in the PNA/DNA duplexes is different from that of a DNA/DNA duplex. This might be due to the different base geometry in a helix which may potentially make hydrogen bonds in a base pair and stacking interaction unfavorable compared with DNA/DNA duplexes.     

In Search of a Hoogsteen Base Paired DNA Duplex in Aqueous Solution

Abstract

When the oligodeoxynucleotides d(A)₆ and d(T)₆ are mixed together in a 1:1 ratio (in 100 mM NaCl), the NH signals in the NMR spectrum gave a typical signature of Watson-Crick paired (WC) and Hoogsteen paired (H) AT base pairs. The observation indicates two schemes: Scheme I, WC and H duplexes in slow equilibrium, i.e., WC ? H, Scheme II, the WC helix formed is unstable and that it disproportionates into a triple helix (TR) and free d(A)₆. We show that (i) addition of extra d(A)₆ does not change the helix composition, (ii) addition of a minor-groove specific drug Dst2 (a distamycin analogue) results in an exclusive WC helix- drug duplex, while it does not destabilize triple helix in a 1:2 mixture. In addition we have compared the melting profile, ³¹P NMR spectra, ¹H NMR spectra and the salt dependence of the 1:1 mixture and that of a pure triple helix. All the data from the above experiments overwhelmingly favor Scheme I. However Scheme II cannot be categorically excluded.

Based on 1D/2D NMR studies, we have characterized the structural properties of the Hoogsteen double helix in terms of nucleotide conformations. In addition, we computationally demonstrate that the relative stability of the WC over the H duplexes increases with increasing chain length.     

Solution structure of a DNA duplex containing 8-hydroxy-2'-deoxyguanosine opposite deoxyguanosine

Deoxyguanosine residues are hydroxylated by reactive oxygen species at the C-8 position to form 8-hydroxy-2'-deoxyguanosine (8-OG), one of the most important mutagenic lesions in DNA. Though the spontaneous G:C to C:G transversions are rare events, the pathways leading to this mutation are not established. An 8-OG:G mispair, if not corrected by DNA repair enzymes, could lead to G:C to C:G transversions. NMR spectroscopy and restrained molecular dynamics calculations are used to refine the solution structure of the base mismatch formed by the 8-OG:G pair on a self complementary DNA dodecamer duplex d(CGCGAATT(8-O)GGCG)(2). The results reveal that the 8-OG base is inserted into the helix and forms Hoogsteen base-pairing with the G on the opposite strand. The 8-OG:G base-pairs are seen to be stabilized by two hydrogen bonding interactions, one between the H7 of the 8-OG and the O6 of the G, and a three-center hydrogen bonding between the O8 of the 8-OG and the imino and amino protons of the G. The 8-OG:G base-pairs are very well stacked between the Watson-Crick base-paired flanking bases. Both strands of the DNA duplex adopt right-handed conformations. All of the unmodified bases, including the G at the lesion site, adopt anti glycosidic torsion angles and form Watson-Crick base-pairs. At the lesion site, the 8-OG residues adopt syn conformations. The structural studies demonstrate that 8-OG(syn):G(anti) forms a stable pair in the interior of the duplex, providing a basis for the in vivo incorporation of G opposite 8-OG. Calculated helical parameters and backbone torsional angles, and the observed 31P chemical shifts, indicate that the structure of the duplex is perturbed near lesion sites, with the local unwinding of the double helix. The melting temperature of the 8-OG:G containing duplex is only 2.6 deg. C less than the t(m) of the unmodified duplex.     

Error‐free and mutagenic processing of topoisomerase 1‐provoked damage at genomic ribonucleotides

Genomic ribonucleotides incorporated during DNA replication are commonly repaired by RNase H2‐dependent ribonucleotide excision repair (RER). When RNase H2 is compromised, such as in Aicardi‐Goutières patients, genomic ribonucleotides either persist or are processed by DNA topoisomerase 1 (Top1) by either error‐free or mutagenic repair. Here, we present a biochemical analysis of these pathways. Top1 cleavage at genomic ribonucleotides can produce ribonucleoside‐2′,3′‐cyclic phosphate‐terminated nicks. Remarkably, this nick is rapidly reverted by Top1, thereby providing another opportunity for repair by RER. However, the 2′,3′‐cyclic phosphate‐terminated nick is also processed by Top1 incision, generally 2 nucleotides upstream of the nick, which produces a covalent Top1–DNA complex with a 2‐nucleotide gap. We show that these covalent complexes can be processed by proteolysis, followed by removal of the phospho‐peptide by Tdp1 and the 3′‐phosphate by Tpp1 to mediate error‐free repair. However, when the 2‐nucleotide gap is associated with a dinucleotide repeat sequence, sequence slippage re‐alignment followed by Top1‐mediated religation can occur which results in 2‐nucleotide deletion. The efficiency of deletion formation shows strong sequence‐context dependence.     

Prediction of atomic structure from sequence for double helical DNA oligomers

DNA can adopt different conformations depending on the base sequence, solvent, electrolyte composition and concentration, pH, temperature, and interaction with proteins. Here we present a model for calculating the three-dimensional atomic structure of double-stranded DNA oligomers. A theoretical energy function is used for calculating the interactions within the base steps and an empirical backbone function is used to restrict the conformational space accessible to the bases and to account for the conformational coupling of neighboring steps in a sequence. Conformational searching on large structures or a large number of structures is possible, because each base step can be described by just two primary degrees of freedom (slide and shift). A genetic algorithm is used to search for low-energy structures in slide-shift space, and this allows very rapid optimization of DNA oligomers. The other base step parameters have been previously optimized for all possible slide-shift sequence combinations, and a heuristic algorithm is used to add the atomic details of the backbone conformation in the final step of the calculation. The structures obtained by this method are very similar to the corresponding X-ray crystal structures observed experimentally. The average RMSD is 2.24 Angstroms for a set of 20 oligomer structures. For 15 of these sequences, the X-ray crystal structure is the global energy minimum. The other 5 are bistable sequences that have B-form global energy minima but crystallize as A-DNA.