Identification of I:A mismatch base-pairing structure in DNA

Deoxyoligonucleotides containing deoxyinosine residues at positions corresponding to ambiguous nucleotides derived from an amino acid sequence have been successfully used as hybridization probes. It is assumed that the hypoxanthine residue can make base pairs with multiple bases. In order to obtain direct evidence for I:A base-pairing, a self-complementary deoxyoligonucleotide, d(G-G-I-A-C-C), was synthesized and its properties were examined by NMR spectroscopy. Three hydrogen-bonded imino proton resonances are observed at low temperatures in H2O suggesting the formation of a self-duplex with complete base pairing. Nuclear Overhauser effect (NOE) experiments showed that a signal at 15.1 ppm originated from the imino proton (H1) of the dI residue (I3) which is hydrogen-bonded to the dA residue (A4). Both the I3 and A4 residues were assumed to have taken an anti glycosidic conformation since irradiating the H1 of I3 gave NOEs both to its own H2 and to that of A4, an NOE also being observed between the H2 protons of I3 and A4. Comparison of the 31P NMR spectra of d(G-G-I-A-C-C) and d(G-G-I-C-C-C) showed the backbone structure of d(G-G-I-A-C-C) to have been disturbed by the presence of purine:purine base pairs in the middle of the hexamer duplex.     

Duplex-hairpin transitions in DNA: NMR studies on CGCGTATACGCG.

Two dimensional NMR methods have been used to assign proton resonances in the high salt (greater than or equal to 100mM Na+), low temperature duplex form of the self-complementary DNA dodecamer d(CGCGTATACGCG). At low salt (less than or equal to 10mM Na+) and higher temperature marked changes in the two-dimensional spectrum, and in the one-dimensional spectrum reported by others, indicate that the molecule converts to an alternate conformation. Using saturation transfer methods, many of the resonances of this new conformation have been assigned, and the kinetics of the interconversion of the two forms has been studied. The linewidth, correlation time, and concentration dependence of the formation of this alternate conformation support the idea that it is a unimolecular hairpin. Observation of chemical shifts and NOEs in the hairpin conformation allow some preliminary structural characterization. Examination of the energetics of the interconversion suggests that the exchange between forms does not proceed through a single stranded intermediate, but rather through another pathway, probably involving a cruciform structure.     

Single molecule studies of DNA mismatch repair

DNA mismatch repair, which involves is a widely conserved set of proteins, is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair.     

Influence of flanking sequence context on the conformational flexibility of aminofluorene-modified dG adduct in dA mismatch DNA duplexes

When positioned opposite to a dA in a DNA duplex, the prototype arylamine–DNA adduct [N-(2′-deoxyguanosin-yl)-7-fluoro-2-aminofluorene (FAF)] adopts the so-called ‘wedge’ (W) conformation, in which the carcinogen resides in the minor groove of the duplex. All 16 FAF-modified 12-mer NG*N/NAN dA mismatch duplexes (G* = FAF, N = G, A, C, T) exhibited strongly positive induced circular dichroism in the 290–360 nm range (ICD_{290–360 nm}), which supports the W conformation. The ICD_{290–360 nm} intensities were the greatest for duplexes with a 3′-flanking T. The AG*N duplex series showed little adduct-induced destabilization. An exception was the AG*T duplex, which displayed two well-resolved signals in the ¹⁹F NMR spectra. This was presumably due to a strong lesion-destabilizing effect of the 3′-T. The flanking T effect was substantiated further by findings with the TG*T duplex, which exhibited greater lesion flexibility and nucleotide excision repair recognition. Adduct conformational heterogeneity decreased in order of TG*T > AG*T > CG*T > AG*A > AG*G > AG*C. The dramatic flanking T effect on W-conformeric duplexes is consistent with the strong dependence of the ICD_290-360 on both temperature and salt concentration and could be extended to the arylamine food mutagens that are biologically relevant in humans.     

31P NMR studies of the solution structure and dynamics of nucleosomes and DNA.

31P NMR studies of 140 base pair DNA fragments in nucleosomes and free in solution show no detectable change in the internucleotide 31P chemical shift or linewidth when DNA is packaged into nucleosomes. Measurements of 31P spin-lattice relaxation times T1 and 31P-[H] nuclear Overhauser enhancements revealed internal motion with a correlation time of about 4 x 10(-10) sec in double helical DNA, both free in solution and bound to nucleosomal core proteins. This result implies greater dynamic mobility in double helical DNA than has previously been supposed.     

In vitro studies of DNA mismatch repair proteins

The ability to monitor and characterize DNA mismatch repair activity in various mammalian cells is important for understanding mechanisms involved in mutagenesis and tumorigenesis. Since mismatch repair proteins recognize mismatches containing both normal and chemically altered or damaged bases, in vitro assays must accommodate a variety of mismatches in different sequence contexts. Here we describe the construction of DNA mismatch substrates containing G:T or O⁶meG:T mismatches, the purification of recombinant native human MutSα (MSH2–MSH6) and MutLα (MLH1–PMS2) proteins, and in vitro mismatch repair and excision assays that can be adapted to study mismatch repair in nuclear extracts from mismatch repair proficient and deficient cells.     

The molecular electrostatic potential and steric accessibility of poly (dI.dC). Comparison with poly (dG.dC)

The influence of the amino group of guanine on the molecular electrostatic potential and the accessibility to reactive sites of B-DNA is investigated by comparing the two model double helices poly (dI.dC) and poly (dG.dC). The calculations clarify the “disruptive” role of the guanine amino group on nucleic acid-polypeptide interactions.     

NMR studies of molecular structure in fruit cuticle polyesters.

The cuticle of higher plants functions primarily as a protective barrier for the leaves and fruits, controlling microbial attack as well as the diffusion of water and chemicals from the outside environment. Its major chemical constituents are waxes (for waterproofing) and cutin (a structural support polymer). However, the insolubility of cutin has hampered investigations of its covalent structure and domain architecture, which are viewed as essential for the design of crop protection strategies and the development of improved synthetic waterproofing materials. Recently developed strategies designed to meet these investigative challenges include partial depolymerization using enzymatic or chemical reagents and spectroscopic examination of the intact polyesters in a solvent-swelled form. The soluble oligomers from degradative treatments of lime fruit cutin are composed primarily of the expected 10,16-dihydroxyhexadecanoic and 16-hydroxy-10-oxo-hexadecanoic acids; low-temperature HF treatments also reveal sugar units that are covalently attached to the hydroxyfatty acids. Parallel investigations of solvent-swollen cutin using 2D NMR spectroscopy assisted by magic-angle spinning yield well-resolved spectra that permit detailed comparisons to be made among chemical moieties present in the intact biopolymer, the soluble degradation products, and the unreacted solid residue.     

1H NMR studies of lac-operator DNA fragments.

The hydrogen-bonded imino protons of a 14 base pair double-stranded DNA fragment comprising one half of the lac operator of E. coli were investigated by 360 MHz H NMR. From combined melting studies of this synthetic 14 b.p. fragment and its two constituent 7 b.p. fragments a nearly complete assignment for the low-field proton resonances was obtained. The experimental spectra are compared with calculated spectra and with the spectrum of a 51 b.p. DNA restriction fragment from E. coli containing the complete lac operator. Structural information on these oligonucleotides is presented. This study is a prerequisite for future 1H NMR investigations of the interaction of the lac operator with the lac repressor.     

NMR studies of a DNA containing 8-hydroxydeoxyguanosine.

The effects of hydroxylation at the C8 of a deoxyguanosine residue in DNA were studied by NMR analysis of a self-complementary dodecanucleotide, d(C1-G2-C3-oh8G4-A5-A6-T7-T8-C9-G10-C11-G12), which has an 8-hydroxy-2'-deoxyguanosine (oh8dG) residue at the 4th position. NMR data indicate that the 8-hydroxyguanine (oh8G) base takes a 6,8-diketo tautomeric form and is base-paired to C with Watson-Crick type hydrogen bonds in a B-form structure. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(CGCGAATTCGCG) duplex. The structural changes caused by 8-hydroxylation of the deoxyguanosine, if any, are localized near the modification site.     

Statistical significance of partial base-pairing potential: implications for recombination of SV40 DNA in eukaryotic cells.

The frequent detection of "patchy homology" between recombining DNAs in eukaryotic systems suggests that partial sequence homology might facilitate formation of partially base-paired hybrid structures and thus define a specific target for recombination. Indeed, the extent of such "patchy homology" initially appears impressive. The question of whether such homology is statistically significant, however, has not been addressed. In this paper we compare the extent of "patchy homology" within the sequence of SV40 recombination sites and within randomly-generated sequences with the same average GC content. We have found no statistically significant differences favoring the existence of "patchy homology" within the recombining regions of SV40. On the average 50% of the bases in randomly-generated sequences with the same GC content can be paired in a pattern of "patchy homology". We have also assessed the ability of sequences near recombination junctions in SV40 to form partially base-paired intra-strand secondary structures. Again, the ability of these sequences to form such configurations was unremarkable when compared with random sequences. Thus, the notion that partial base-pairing provides specificity for sites of recombination must be considered with caution.     

Very stable mismatch duplexes: structural and thermodynamic studies on tandem G.A mismatches in DNA.

We have used ultraviolet melting techniques to compare the stability of several DNA duplexes containing tandem G.A mismatches to similar duplexes containing tandem A.G, I.A, and T.A base pairs. We have found that tandem G.A mismatches in 5'-Y-G-A-R-3' duplexes are more stable than their I.A counterparts and that they are sometimes more stable than tandem 5'-Y-T-A-R-3' sequences. This is not the case for tandem G.A mismatches in other base stacking environments, and it suggests that tandem G.A mismatches in 5'-Y-G-A-R-3' sequences have a unique configuration. In contrast to tandem 5'-G-A-3' mismatches, tandem 5'-A-G-3' mismatches were found to be unstable in all cases examined.     

NMR studies of lantibiotics. The structure of nisin in aqueous solution.

Nisin is a posttranslationally modified protein of 34 amino acids, and is a member of the class of bacteriocidal polypeptides known as lantibiotics, that contain the unusual amino acid lanthionine. Its structure in aqueous solution has been determined on the basis of NMR data, i.e. interproton distance constraints derived from nuclear Overhauser enhancement spectroscopy and torsion angle constraints derived from double-quantum-filtered correlated spectroscopy. Translation of the NMR constraints into a three-dimensional structure was carried out with the distance-geometry program DISMAN, followed by restrained energy minimization using CHARMm. The internal mobility of the peptide chain prohibited the determination of a precise overall folding of the molecule, but parts of the structure could be obtained, albeit sometimes with low resolution. The structure of nisin can best be defined as follows. The outermost N-terminal and C-terminal regions of nisin appear quite flexible, the remainder of the molecule consists of an amphiphilic N-terminal fragment (residues 3-19), joined by a flexible 'hinge' region to a rigid double-ring fragment formed by residues 23-28. The latter fragment has the appearance of a somewhat overwound alpha-helix. It is suggested, by assuming the presence of a (transient) alpha-helical structure in this part of prenisin, that the coupling between residues 23 and 26, as well as between 25 and 28, by thioether bridges, and the inversion of the C alpha chiralities at positions 23 and 25, can be rationalized.     

Solution structure of an oncogenic DNA duplex containing a G.A mismatch

The DNA duplex 5'-d(GCCACAAGCTC).d(GAGCTGGTGGC), which contains a central G.A mismatch has been studied by one and two-dimensional NMR techniques. The duplex corresponds to the sequence 29-39 of the K-ras gene. The mismatch position is that of the first base of the Gly12 codon, a hot spot for mutations. The observed NOEs of the nonexchangeable protons show that both of the bases of the mismatched pair are intrahelical over a wide range of pH. However, the structure of the G.A mispair and the conformation of the central part of the duplex change with pH. This structural change shows a pK of 6.0. At low pH, the G.A bases are base paired with hydrogen bonds between the keto group of the G residue and the amino group of the A residue and, secondly, between the N7 of the G and a proton on N1 of A. This causes the G residue to adopt a syn conformation. On raising the pH, the N1-H proton of the protonated A residue is removed, and the base pair rearranges. In the neutral G.A base pair both residues adopt an anti conformation, and the mismatch is stabilized by hydrogen bonds. Our results on the exchangeable and A(H2) protons of the mismatched pair indicate a shift from a classical face-to-face two hydrogen-bonded structure to a slipped structure stabilized by bifurcated hydrogen bonds. This may be a particular characteristics of this oncogenic sequence in which the G.A error is poorly repaired.     

Multinuclear NMR studies of DNA hairpins. 2. Sequence-dependent structural variations

The solution conformation of three related DNA hairpins, each with five bases in the loop, is investigated by proton and phosphorus 2D NMR methods. The sequences of the three oligomers are d(CGCGTTGTTCGCG), d(CGCGTTTGTCGCG), and d(CTGCTCTTGTTGAGCAG). One pair of hairpins shares the same stem sequence but differs in the loop, and the appearance of an unusual phosphate torsion in the stem is found to depend on the sequence in the loop of the hairpin. The second pair of hairpins shares the same loop region but differs in the stem sequence in that the base pair which closes the loop is a C-G or G-C pair. The pattern of NOEs reveals that the stacking arrangement in the loop region depends on the base pair that closes the stem. These results suggest that hairpin loop conformation and dynamics are sensitive to small changes in the loop and adjacent stem sequences. These findings are discussed in relation to sequence-dependent thermodynamic changes that have been observed in RNA hairpins.     

Maternal effect for DNA mismatch repair in the mouse.

DNA mismatch repair (DMR) functions to maintain genome stability. Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences. Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of approximately 10% was observed. To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F(1) progeny from Pms2(-/-) female mice mated with wild-type males. Our analysis indicates that MSI in the female germ line was approximately 9%. MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient. We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions. The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote. The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene.