Theoretical Investigation of the Molecular Structure of the πκ DNA Base Pair

Abstract

The structure of the nonclassical πκ base pair (7–methyl-oxoformycin … 2,4-diaminopyrimidine) was studied at the ab initio Hartree-Fock (HF) and MP2 levels using the 6–31G* and 6–31G** basis sets. The πκ base pair is bound by three parallel hydrogen bonds with the donor-acceptor-donor recognition pattern. Recently, these bases were proposed as an extension of the genetic alphabet from four to six letters (Piccirilli et al. Nature 343, 33(1990)). By the HF/6- 31G* method with full geometry optimization we calculated the 12 degree propeller twist for the minimum energy structure of this complex. The linearity of hydrogen bonds is preserved in the twisted structure by virtue of the pyramidal arrangement of the κ-base amino groups. The rings of both the π and κ molecules remain nearly planar. This nonplanar structure of the πκ base pair is only 0.1 kcal/mol more stable than the planar (Cs) conformation. The HF/6- 31G* level gas-phase interaction energy of πκ (—13.5 kcal/mol) calculated by us turned out to be nearly the same as the interaction energy obtained previously for the adenine-thymine base pair (—13.4 kcal/mol) at the same computational level. The inclusion of p-polarization functions on hydrogens, electron correlation effects (MP2/6–31G** level), and the correction for the basis set superposition error (BSSE) increase this energy to -14.0 kcal/mol.     

A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs

Abstract

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. We have substituted four dA · dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1 · D2) with dG · dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG · dC base pairs (ps-D5 · D6) is 10-16°C lower and the van't Hoff enthalpy difference Δ_vH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl₂) compared to that of ps-Dl · D2. Based on energy minimizations of a ps-[d(T₅GA₅) · d(A₅CT₅)] duplex using force field calculations we propose a model for the conformation of a trans dG · dC base pair in a ps helix.     

The in situ formation of DNA · DNA duplexes of Drosophila virilis satellite DNA

The DNA of Drosophila virilis brains and imaginal discs was labeled in vitro to a specific activity of 6 X 10(-5) dpm/mug, using an organ culture medium. The DNA was fractionated on neutral and alkaline CsC1 gradients and the heavy strands of satellite I annealed in situ to denatured polytene chromosomes from squash preparations of larval salivary glands. Nuclease S1 from Aspergillus oryzae was used to digest the unpaired ssDNA, resulting in a distinct labeling of the alpha-heterochromatin in the chromocenter and a small amount of diffused labeling in the proximal beta-heterochromatic part of the X-Chromsome.     

Protein–Protein Interactions in DNA Base Excision Repair

The system of base excision repair (BER) ensures correction of the most abundant DNA damages in mammalian cells and plays an important role in maintaining genome stability. Enzymes and protein factors participate in the multistage BER in a coordinated fashion, which ensures repair efficiency. The suggested coordination mechanisms are based on formation of protein complexes stabilized via either direct or indirect DNA-mediated interactions. The results of investigation of direct interactions of the proteins participating in BER with each other and with other proteins are outlined in this review. The known protein partners and sites responsible for their interaction are presented for the main participants as well as quantitative characteristics of their affinity. Information on the mechanisms of regulation of protein–protein interactions mediated by DNA intermediates and posttranslational modification is presented. It can be suggested based on all available data that the multiprotein complexes are formed on chromatin independent of the DNA damage with the help of key regulators of the BER process – scaffold protein XRCC1 and poly(ADP-ribose) polymerase 1. The composition of multiprotein complexes changes dynamically depending on the DNA damage and the stage of BER process.     

Flexibility of A · Pairs in Left-Handed DNA Helices

Abstract

We have analysed by various approaches the structure of cloned synthetic sequences in supercoiled plasmids. Individual inserts were formed by d(C-G)_n blocks interrupted by the presence of A · T pairs positioned either in phase or out of phase of pur-pyr alternation. Based on the thermodynamic analysis we obtained results confirming that A · T pairs are easily incorporated into left-handed helices without significant energetic penalty. Sequences GTAC which are known to form cruciform structures in multiple repetition underwent a B-Z transition. In the case of plasmids containing AA/TT code words and substantial discontinuities in purine-pyrimidine alternation our analysis indicates that Z-Z junctions formed by A · T pairs contributed little to the overall energetic demands of the B-Z transition probably thanks to their high conformational flexibility.     

The Essential Role of the 3′ Terminal Template Base in the First Steps of Protein-Primed DNA Replication

Bacteriophages φ29 and Nf from Bacillus subtilis start replication of their linear genomes at both ends using a protein-primed mechanism by means of which the DNA polymerase initiates replication by adding dAMP to the terminal protein, this insertion being directed by the second and third 3′ terminal thymine of the template strand, respectively. In this work, we have obtained evidences about the role of the 3′ terminal base during the initiation steps of φ29 and Nf genome replication. The results indicate that the absence of the 3′ terminal base modifies the initiation position carried out by φ29 DNA polymerase in such a way that now the third position of the template, instead of the second one, guides the incorporation of the initiating nucleotide. In the case of Nf, although the lack of the 3′ terminal base has no effect on the initiation position, its absence impairs further elongation of the TP-dAMP initiation product. The results show the essential role of the 3′ terminal base in guaranteeing the correct positioning of replication origins at the polymerization active site to allow accurate initiation of replication and further elongation.     

The Concerted Action of Msh2 and UNG Stimulates Somatic Hypermutation at A · T Base Pairs

Mismatch repair plays an essential role in reducing the cellular mutation load. Paradoxically, proteins in this pathway produce A·T mutations during the somatic hypermutation of immunoglobulin genes. Although recent evidence implicates the translesional DNA polymerase η in producing these mutations, it is unknown how this or other translesional polymerases are recruited to immunoglobulin genes, since these enzymes are not normally utilized in conventional mismatch repair. In this report, we demonstrate that A·T mutations were closely associated with transversion mutations at a deoxycytidine. Furthermore, deficiency in uracil-N-glycolase (UNG) or mismatch repair reduced this association. These data reveal a previously unknown interaction between the base excision and mismatch repair pathways and indicate that an abasic site generated by UNG within the mismatch repair tract recruits an error-prone polymerase, which then introduces A·T mutations. Our analysis further indicates that repair tracts typically are ∼200 nucleotides long and that polymerase η makes ∼1 error per 300 T nucleotides. The concerted action of Msh2 and UNG in stimulating A·T mutations also may have implications for mutagenesis at sites of spontaneous cytidine deamination.The affinity maturation of the antibody response depends on the somatic hypermutation (SHM) process. The enzyme activation-induced cytidine deaminase (AID) initiates SHM in germinal center B cells by deaminating C within immunoglobulin (Ig) genes, yielding a G·U lesion that is resolved by several mechanisms (29). Replication across the U generates G·C to A·T transition mutations, while the removal of the U by uracil-N-glycolase (UNG) leads to transversion and transition mutations at the original G·C base pair (33). The AID-generated G·U lesion is also a substrate for the mismatch repair (MMR) proteins Msh2, Msh6, and Exo1. Unlike their normal role in DNA repair, the processing of this lesion by these MMR proteins during SHM paradoxically leads to the production of mutations at A·T base pairs (see below).MMR is a DNA repair process utilized by prokaryotes and eukaryotes (25). This pathway repairs DNA errors caused by the misincorporation of nucleotides during DNA synthesis. The initial mismatch is detected by MutSα, which consists of Msh2 and Msh6 in mammalian cells. The ability of MMR to discriminate between the mutated and unmutated strands of DNA is thought to be dictated by nicks or gaps on the newly synthesized lagging strand between Okazaki fragments or by strand ends on the leading strand at the replication fork (18). The MutLα endonuclease (Mlh1/Pms2) uses the DNA nick or end as a marker of the newly synthesized, and therefore mutated, strand to introduce a new nick on either side of the mismatch (15). This nicked strand is then excised by the 5′-to-3′ exonuclease Exo1, and the ensuing gap is repaired by the replicative polymerase δ. However, since AID acts primarily during G₁ of the cell cycle (11, 36), it is unclear whether Msh2/6 is capable of distinguishing between the AID-mutated and unmutated strands prior to strand excision.Consistently with their role in DNA repair, deficiency in Msh2, Msh6, or Exo1 generally leads to an increase in mutation frequencies in different tissues (40). However, in the case of SHM of Ig genes, the loss of these MMR proteins reduces the frequency of mutations at A·T base pairs (4, 5, 10, 16, 22, 30, 32, 37, 41, 42). One possible difference between conventional and mutagenic MMR is the involvement of the error-prone DNA polymerase η in the latter process. Indeed, both mice and humans lacking polymerase η resemble Msh2-deficient mice, in that mutations at A·T base pairs in the V region are less frequent (6, 7, 47). Moreover, the error spectrum of polymerase η on undamaged DNA matches the mutation spectrum of A·T mutations in the V region (35). While it is now well established that mutations at A·T base pairs are produced largely by proteins involved in the MMR pathway, it is not known how DNA polymerase η is recruited during SHM.One possible explanation for the use of error-prone polymerases is the occurrence of replication-blocking lesions, such as an abasic site or a modified nucleotide, in the V region of Ig genes. Evidence that a replication block leads to mutagenic MMR comes from recent studies showing the requirement of ubiquitinated PCNA for mutagenic MMR (1, 19, 34). Monoubiquitination at the K164 residue of PCNA in response to DNA damage leads to translesional synthesis (1), and SHM at A·T base pairs is reduced in PCNA^K164R/K164R mice to levels observed in MMR-deficient mice (19, 34). In addition, the finding that translesional DNA polymerases are involved in SHM (9, 31, 45-47) suggests that replication-blocking lesions are common at the Ig locus during SHM. Taken together, these observations suggest a model in which replication-blocking lesions recruit error-prone polymerases, which then generate mutations at nearby A·T base pairs. As reported here, we have tested this model by examining the correlated mutations in V region sequences from hypermutating Ramos cells and in murine centroblasts.     

Base recognition of gap sites in DNA–DNA and DNA–RNA duplexes by short oligonucleotides

To increase base recognition capability and sensitivity, we propose the separation of a commonly used single-probe system for oligonucleotide analysis into a set of three probes: a fluorophore-labeled probe, a promoter probe, and a short probe. In this study, we found that the probes of only 4 nt in length can selectively bind the corresponding gap site on complexes consisting of the target, fluorophore-labeled probe, and promoter probe, exhibiting a more than 14-fold difference in ligation between the matched and mismatched sequences. Moreover, we demonstrated that the immobilized short probes accurately recognized the sequences of the gap sites.     

The physicochemical essence of the purine·pyrimidine transition mismatches with Watson-Crick geometry in DNA: A·C* versa A*·C. A QM and QTAIM atomistic understanding

It was established for the first time by DFT and MP2 quantum-mechanical (QM) methods either in vacuum, so in the continuum with a low dielectric constant (ε = 4), typical for hydrophobic interfaces of specific protein-nucleic acid interactions, that the repertoire for the tautomerisation of the biologically important adenine·cytosine* (A·C*) mismatched DNA base pair, formed by the amino tautomer of the A and the imino mutagenic tautomer of the C, into the A*·C base mispair (?G = 2.72 kcal?mol^?1 obtained at the MP2 level of QM theory in the continuum with ε = 4), formed by the imino mutagenic tautomer of the A and the amino tautomer of the C, proceeds via the asynchronous concerted double proton transfer along two antiparallel H-bonds through the transition state (TS_A·C*?A*·C). The limiting stage of the A·C*→A*·C tautomerisation is the final proton transfer along the intermolecular N6H···N4 H-bond. It was found that the A·C*/A*·C DNA base mispairs with Watson–Crick geometry are associated by the N6H?N4/N4H?N6, N3H?N1/N1H?N3 and C2H?O2 H-bonds, respectively, while the TS_A·C*?A*·C is joined by the N6–H–N4 covalent bridge and the N1H?N3 and C2H?O2 H-bonds. It was revealed that the A·C*?A*·C tautomerisation is assisted by the true C2H?O2 H-bond, that in contrast to the two others conventional H-bonds exists along the entire intrinsic reaction coordinate (IRC) range herewith becoming stronger at the transition from vacuum to the continuum with ε = 4. To better understand the behavior of the intermolecular H-bonds and base mispairs along the IRC of the A·C*?A*·C tautomerisation, the profiles of their electron-topological, energetical, geometrical, polar and charge characteristics are reported in this study. It was established based on the profiles of the H-bond energies that all three H-bonds are cooperative, mutually strengthening each other. The nine key points, providing a detailed physicochemical picture of the A·C*?A*·C tautomerisation, were revealed and thoroughly examined along the IRC. It was shown that the A*·C base mispair with the population ~1 % obtained at the MP2 level of QM theory in the continuum with ε = 4 is thermodynamically and dynamically stable structure. Its lifetime was calculated to be 5.76·10^?10 s at the MP2 level of QM theory in the continuum with ε = 4. This lifetime, from the one side, enables all six low-frequency intermolecular vibrations to develop, but, from the other side, it is by order less than the time (several ns) required for the replication machinery to forcibly dissociate a base pair into the monomers during DNA replication. This means that the A*·C base mispair “slips away from the hands” of the replication machinery into the A·C* mismatched base pair. Consequently, the authors came to the conclusion that exactly the A·C* base mispair is an active player of the point mutational events and is effectively dissociated by the replication machinery into the A and C* monomers in contrast to the A*·C base mispair, playing the mediated role of a provider of the A·C* base mispair in DNA that is synthesised.     

Fluorescence analysis of G·C versus A·T binding of quinacrine to DNA

The affinity of quinacrine for native DNA has been determined from fluorescence measurements and equilibrium dialysis in Tris-HC10.05 m, NaCl0.1 m, EDTA 10^?3m, pH 7.5. When considering M. lysodeiktikus, E. coli calf thymus and C. perfringens the affinities of DNA for quaniactive have been found to change by a factor of two and the fluorescence intensities to change by a factor of 25. The varying affinities and fluoroescence intensities of bound quinacrine are attributed to heterogeneous binding. For all DNAs we have assumed that there exist three classes of intercalation sites: I, A·T-A·T; 2, G·C-G·C; and 3, A·T-G·C, assuming that base pair ordering is less relevant than base composition of sites. By fitting the affinities of native DNAs with this model it was found that quinacrine binds to site 2 three times more strongly than it does to site 1. When flucrescence intensity is studied, triplets of A·T pairs appear to be responsible for the high quantum yield of A·T rich DNA whereas the quenching properties of a G·C base pair adjacent to an intercalated quinacrine are well known.     

The DNA–DNA spacing in gemini surfactants–DOPE–DNA complexes

Gemini surfactants from the homologous series of alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, number of spacer carbons n = 2 – 12) and dioleoylphosphatidylethanolamine (DOPE) were used for cationic liposome (CL) preparation. CLs condense highly polymerized DNA creating complexes. Small-angle X-ray diffraction identified them as condensed lamellar phase L_α^C in the studied range of molar ratios CnGS12/DOPE in the temperature range 20 – 60 °C. The DNA–DNA distance (d_DNA) is studied in dependence to CnGS12 spacer length and membrane surface charge density. The high membrane surface charge densities (CnGS12/DOPE = 0.35 and 0.4 mol/mol) lead to the linear dependence of d_DNA vs. n correlating with the interfacial area of the CnGS12 molecule.     

线粒体DNA G7444A突变可能影响A1555G突变的表型表达

线粒体12S rRNA和tRNASer(UCN) 基因是导致非综合征型听力损失的两个突变热点区域。作者收集了1个母系遗传感音神经性聋家系, 该家系同时携带线粒体DNA (mtDNA) A1555G和G7444A突变。临床资料分析表明, 该家系包括药物致聋的耳聋外显率(所有耳聋患者/所有母系成员)为58%, 而非药物致聋的耳聋外显率(非药物性聋患者/所有母系成员)为25%, 明显高于其他携带A1555G突变的耳聋家系。先证者的线粒体全序列分析表明, 该线粒体基因组共有28个多态位点, 属于东亚人群B4c1单体型。在这些多态位点中, 除A1555G和G7444A突变外, 未发现其他有功能意义的突变。这表明mtDNA G7444A突变可能加重由A1555G突变造成的线粒体功能缺失, 从而增加耳聋的外显率。     

Inactivation of OGG1 increases the incidence of G · C→T · A transversions in Saccharomyces cerevisiae : evidence for endogenous oxidative damage to DNA in eukaryotic cells

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. To investigate the biological role of the OGG1 gene, mutants were constructed by partial deletion of the coding sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1::URA3 mutant strains. The disruption of the OGG1 gene does not compromise the viability of haploid cells, therefore it is not an essential gene. The capacity to repair 8-OxoG has been measured in cell-free extracts of wild-type and ogg1 strains using a 34mer DNA fragment containing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a substrate. Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex. In contrast, cell-free extracts of the Ogg1-deficient strain have no detectable activity that can cleave the 8-OxoG/C duplex. The biological properties of the ogg1 mutant have also been investigated. The results show that the ogg1 disruptant is not hypersensitive to DNA-damaging agents such as ultraviolet light at 254?nm, hydrogen peroxide or methyl methanesulfonate. However, the ogg1 mutant exhibits a mutator phenotype. When compared to those of a wild-type strain, the frequencies of mutation to canavanine resistance (Can^R) and reversion to Lys⁺ are sevenfold and tenfold higher for the ogg1 mutant strain, respectively. Moreover, using a specific tester system, we show that the Ogg1-deficient strain displays a 50-fold increase in spontaneously occurring G?·?C→T?·?A transversions compared to the wild-type strain. The five other base substitution events are not affected by the disruption of the OGG1 gene. These results strongly suggest that endogeneous reactive oxygen species cause DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 protein contributes to the maintenance of genetic stability in S. cerevisiae.     

DNA Polymerases β and λ Mediate Overlapping and Independent Roles in Base Excision Repair in Mouse Embryonic Fibroblasts

Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol β) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol λ), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases λ and β in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol β and pol λ. Neutral red viability assays demonstrated that pol λ and pol β double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol λ to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol λ and pol β interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.     

Chain length-dependent association of distamycin-type oligopeptides with A·T and G·C pairs in polydeoxynucleotide duplexes

Different binding affinities of various distamycin analogs including the deformylated derivative with poly(dA-dC)·poly(dG-dT) were investigated using CD measurements. The inhibitory effect of distamycins on the DNAase I cleavage activity of DNA duplexes strongly supports the binding data. The base specificity of the ligand interaction with duplex DNA depends on the chain length of distamycin analogs. Netropsin, distamycin-2 and the deformylated distamycin-3 show no binding to dG·dC containing sequences at moderate ionic strength and are classified as highly dA·dT specific. In contrast distamycin having three, four or five methylpyrrolecarboxamide groups also forms more or less stable complexes with dG·dC-containing duplexes. These ligands possess a lower basepair specificity. The correlation between binding behavior and oligopeptide structure shows that presence of the number of hydrogen acceptor and donor sites determines the basepair and sequence specificity. The additional interaction with dG·dC pairs becomes essential when the number of hydrogen acceptor sites exceeds n = 3.     

Stressing-out DNA? The contribution of serine-phosphodiester interactions in catalysis by uracil DNA glycosylase

The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiester backbone of damaged DNA using the hydroxyl side chains of a conserved trio of serine residues, resulting in flipping of the deoxyuridine from the DNA helix into the enzyme active site. We have investigated the energetic role of these serine-phosphodiester interactions using the complementary approaches of crystallography, directed mutagenesis, and stereospecific phosphorothioate substitutions. A new crystal structure of UDG bound to 5'-HO-dUAAp-3' (which lacks the 5' phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, and that the enzyme has undergone the same specific clamping motion that brings key active site groups into position as previously observed in the structures of human UDG bound to large duplex DNA substrates. From this structure, it may be concluded that glycosidic bond cleavage and the induced fit conformational change in UDG can occur without the 5' pinching interaction. The S88A, S189A, and S192G "pinching" mutations exhibit 360-, 80-, and 21-fold damaging effects on k(cat)/K(m), respectively, while the S88A/S189A double mutant exhibits an 8200-fold damaging effect. A free energy analysis of the combined effects of nonbridging phosphorothioate substitution and mutation at these positions reveals the presence of a modest amount of strain energy between the compressed 5' and 3' phosphodiester groups flanking the bound uridine. Overall, these results indicate a role for these serine-phosphodiester interactions in uracil flipping and preorganization of the sugar ring into a reactive conformation. However, in contrast to a recent proposal [Parikh, S. S., et al. (2000) Proc Natl. Acad. Sci. 94, 5083], there is no evidence that conformational strain of the glycosidic bond induced by serine pinching plays a major role in the 10(12)-fold rate enhancement brought about by UDG.