Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae.

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.     

Non-planar structure of nitrous bases and non-coplanarity of Watson-Crick pairs.

We discuss the non-planar structural stability of the NH2-group in formamide, cytosine, adenine, guanine and aniline molecules. Based on the microwave data available on small amino derivatives and on the results of PCILO conformation study it is shown that the slope of the amino group HNH plane to the molecular plane in nitrous bases should be close to 40 degrees. One of the main consequences of the non-planar structure of bases is a comparatively large (approximately equal to 15 degrees) propeller twisting of purine and pyrimidine planes in the complementary adenine-thymine and guanine-cytosine pairs. It is concluded that the non-coplanarity of single Watson-Crick base pairs is their intrinsic property. The specificity of hydrogen bonding in pairs along with stacking is believed to be the original cause of their peculiar packing in crystals and in DNA and RNA structures.     

Base pair buckling can eliminate the interstrand purine clash at the CpG steps in B-DNA caused by the base pair propeller twisting

Results of calculations using various empirical potentials suggest that base pair buckling, which commonly occurs in DNA crystal structures, is sufficient to eliminate the steric clash at CpG steps in B-DNA, originating from the base pair propeller twisting. The buckling is formed by an inclination of cytosines while deviations of guanines from a plane perpendicular to the double helix axis are unfavorable. The buckling is accompanied by an increased vertical separation of the base pair centers but the buckled arrangement of base pairs is at least as stable as when the vertical separation is normal and buckle zero. In addition, room is created by the increased vertical separation for the bases to propeller twist as is observed in DNA crystal structures. Further stabilization of base stacking is introduced into the buckled base pair arrangement by roll opening the base pairs into the double helix minor groove. The roll may lead to the double helix bending and liberation of guanines from the strictly perpendicular orientation to the double helix axis. The liberated guanines further contribute to the base pair buckling and stacking improvement. This work also suggests a characteristic very stable DNA structure promoted by nucleotide sequences in which runs of purines follow runs of pyrimidine bases.     

Spontaneous mutational specificity of drug resistance plasmid pKM101 in Escherichia coli.

Plasmid pKM101 enhances the frequency of spontaneous and ultraviolet light-induced mutations in Escherichia coli and protects the cells against the lethal effects of ultraviolet irradiation. By analyzing reversion patterns of defined trpA alleles, we showed that pKM101 caused all types of spontaneous base-pair substitution mutations with the possible exception of guanine . cytosine leads to adenine. thymine transitions. Neither insertion nor deletion frameshift mutations were enhanced. Transversions were more strongly enhanced than transitions, and adenine . thymine base pairs appeared more susceptible to pKM101 mutator activity than guanine . cytosine base pairs. In addition, there were effects from neighboring base pairs and genetic background that influenced the mutator activity of pKM101.     

Index to Authors,Volume 3

Abstract

Results of calculations using various empirical potentials suggest that base pair buckling, which commonly occurs in DNA crystal structures, is sufficient to eliminate the steric clash at CpG steps in B-DNA, originating from the base pair propeller twisting. The buckling is formed by an inclination of cytosines while deviations of guanines from a plane perpendicular to the double helix axis are unfavorable. The buckling is accompanied by an increased vertical separation of the base pair centers but the buckled arrangement of base pairs is at least as stable as when the vertical separation is normal and buckle zero. In addition, room is created by the increased vertical separation for the bases to propeller twist as is observed in DNA crystal structures. Further stabilization of base stacking is introduced into the buckled base pair arrangement by roll opening the base pairs into the double helix minor groove. The roll may lead to the double helix bending and liberation of guanines from the strictly perpendicular orientation to the double helix axis. The liberated guanines further contribute to the base pair buckling and stacking improvement. This work also suggests a characteristic very stable DNA structure promoted by nucleotide sequences in which runs of purines follow runs of pyrimidine bases.     

Perturbation of hydrogen bonds in the adenine ... thymine base pair by Na+, Mg2+, Ca2+ and NH4+ cations

Perturbation of the hydrogen bonds in the adenine ... thymine base pair by Na+, Mg2+, Ca2+ and NH4+ cations has been investigated by means of ab initio SCF calculations with the STO-3G basis set. The geometry of adenine...thymine, as well as those of the perturbed pairs were optimized. Approach of any cation to thymine at O6 leads to destabilization of the adenine...thymine pair; divalent cations (Mg2+, Ca2+) have a profound effect on the structure of the base pair. The approach of a cation to other available sites (thymine: O2, adenine N1 and N3) leads, on the other hand, to stabilization of the base pair. If a water molecule is placed between the cation and the base pair, the structure and stability of the base pair are changed only negligibly.     

On the hydration of DNA. II. Base composition dependence of the net hydration of DNA

The dependence of the net hydration of DNA on its base composition has been measured by density gradient ultracentrifugation of three DNA's in a series of cesium and lithium salt solutions of different water activities. Extrapolation to zero water activity showed the dependence of the partial specific volume on base composition to be very small for CsDNA and aero for LiDNA. At least 99% of the dependence of buoyant density on base composition can be accounted for on the basis of a differential hydration, with a mole of adenine–thymine pairs binding about 2 moles more water than a mole of guanine–cytosine pairs in CsCl.     

M.HhaI binds tightly to substrates containing mismatches at the target base.

The (cytosine-5) DNA methyltransferase M.HhaI causes its target cytosine base to be flipped completely out of the DNA helix upon binding. We have investigated the effects of replacing the target cytosine by other, mismatched bases, including adenine, guanine, thymine and uracil. We find that M.HhaI binds more tightly to such mismatched substrates and can even transfer a methyl group to uracil if a G:U mismatch is present. Other mismatched substrates in which the orphan guanine is changed exhibit similar behavior. Overall, the affinity of DNA binding correlates inversely with the stability of the target base pair, while the nature of the target base appears irrelevant for complex formation. The presence of a cofactor analog. S-adenosyl-L-homocysteine, greatly enhances the selectivity of the methyltransferase for cytosine at the target site. We propose that the DNA methyltransferases have evolved from mismatch binding proteins and that base flipping was, and still is, a key element in many DNA-enzyme interactions.     

Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy.

Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich.     

基于密度泛函理论研究Watson–Crick碱基对中的氢键特征(英文)

基于密度泛函理论(DFT)研究腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶以及腺嘌呤胸腺嘧啶碱基对、鸟嘌呤胞嘧啶碱基对。在DFT-B3LYP/6-31G**水平上利用自然键轨道理论分析研究结果显示,互补碱基对的结构和电子特征有利于氢键的形成。本文中讨论几何结构、电子结构、分子轨道和能量对于氢键形成的影响。此研究结果将有助于更好的理解AT和GC碱基对中氢键与它们的结构特性之间的关系。     

Molecular recognition of B-DNA by Hoechst 33258.

The binding sites of Hoechst 33258, netropsin and distamycin on three DNA restriction fragments from plasmid pBR322 were compared by footprinting with methidiumpropyl-EDTA X Fe(II) [MPE X Fe(II)]. Hoechst, netropsin and distamycin share common binding sites that are five +/- one bp in size and rich in A X T DNA base pairs. The five base pair protection patterns for Hoechst may result from a central three base pair recognition site bound by two bisbenzimidazole NHs forming a bridge on the floor of the minor groove between adjacent adenine N3 and thymine O2 atoms on opposite helix strands. Hydrophobic interaction of the flanking phenol and N-methylpiperazine rings would afford a steric blockade of one additional base pair on each side.     

Distribution and specificity of mutations induced by neocarzinostatin in the lacI gene of Escherichia coli.

Although neocarzinostatin (NCS) attacks DNA almost exclusively at adenine and thymine residues in vitro, exposure of Escherichia coli to this antitumor drug resulted in a high frequency of mutations at guanine:cytosine base pairs in the lacI gene. Thus, NCS-induced base substitution mutations do not appear to result from the major DNA lesions that have been biochemically characterized. The overall distribution of nonsense mutations produced by NCS was distinctly nonrandom, consisting in part of a few "hotspots" and a large number of "coldspots." The existence of these coldspots implies that untargeted mutagenesis does not make a significant contribution to the mutations induced by this SOS-dependent mutagen.     

Biological consequences of free radical-damaged DNA bases

The principal oxidized cytosine bases, uracil glycol, 5-hydroxycytosine, and 5-hydroxyuracil, are readily bypassed, miscode, and are thus important premutagenic lesions. Similarly the principal oxidation product of guanine, 8-oxoguanine, miscodes with A and is a premutagenic lesion. Most of the thymine and adenine products that retain their ring structure primarily pair with their cognate bases and are not potent premutagenic lesions. Although thymine glycol pairs with its cognate base and is not mutagenic it significantly distorts the DNA molecule and is a lethal lesion. Ring fragmentation, ring contraction, and ring open products of both pyrimidines and purines block DNA polymerases and are potentially lethal lesions. Although these breakdown products have the potential to mispair during translesion synthesis, the mutational spectra of prokaryotic mutants defective in the pyrimidine-specific and/or purine-specific DNA glycosylases do not reflect that expected of the breakdown products. Taken together, the data suggest that the principal biological consequences of endogenously produced and unrepaired free radical-damaged DNA bases are mutations.     

Perturbation of Hydrogen Bonds in the Adenine…Thymine Base Pair by Na+, Mg2+, Ca2+ and NH4 + Cations

Abstract

Perturbation of the hydrogen bonds in the adenine…thymine base pair by Na⁺, Mg²⁺, Ca²⁺ and NH₄ ⁺ cations has been investigated by means of ab initio SCF calculations with the STO-3G basis set. The geometry of adenine…thymine, as well as those of the perturbed pairs were optimized. Approach of any cation to thymine at 06 leads to destabilization of the adenine…thy mine pair; divalent cations (Mg²⁺, Ca²⁺) have a profound effect on the structure of the base pair. The approach of a cation to other available sites (thymine: O2, adenine N1 and N3) leads, on the other hand, to stabilization of the base pair. If a water molecule is placed between the cation and the base pair, the structure and stability of the base pair are changed only negligibly.     

Expanding the Genetic Code: Unnatural Base Pairs in Biological Systems

Molecular Biology - The genetic code is considered to use five nucleic bases (adenine, guanine, cytosine, thymine and uracil), which form two pairs for encoding information in DNA and two pairs for...     

Molecular-Mechanical studies of the mismatched base analogs of d(CGCGAATTCGCG)2:d(CGTGAATTCGCG)2, d(CGAGAATTCGCG)2, d(CGCGAATTCACG)2, d(CGCGAATTCTCG)2, and d(CGCAGAATTCGCG)·d(CGCGAATTCGCG)

Molecular-mechanical simulations have been carried out on “mismatched base” analogs of the DNA double-helical structure d(CGCGAATTCGCG)₂, in which the base pairs CG at the 3 and 10 positions have been replaced by CA, AG, TC, and TG base pairs, as well as an insertion analog in which an extra adenine has been incorporated into one strand of the above structure between bases 3 and 4. The results of these simulations (calculated relative stabilities, structures, and nmr ring-current shifts) have been compared with calorimetric and nmr data. The calculated relative stabilities of the double-helical parent dodecamer and the various “wobble” base pairs qualitatively correlate with the experimental melting temperatures. The base-pairing structure for the GT wobble pair is in agreement with that previously determined from nmr experiments. For the GA base pair, the structure with both bases anti has a slightly more favorable energy from base pairing and stacking than a structure with non-Watson-Crick H-bonding with adenine syn, in agreement with nmr experiments. The CA wobble base is calculated to favor an adenine 6NH₂ …? cytosine N3 H-bond over cytosine 4NH₂ …? adenine N1, again, in agreement with nmr experiments. There is no definitive experimental data on the TC base pair, but the existence of (somewhat long and weak) H-bonds involving cytosine 4NH₂ …? thymine 4CO and cytosine N3 …? thymine HN3 seems reasonable. We find a structure in which the extra adenine base of the insertion analogs sits “inside” the double helix.     

Crystal structure of a Z-DNA fragment containing thymine/2-aminoadenine base pairs

The Z-DNA structure has been shown to form in two crystals made from self-complementary DNA hexamers d(CGTDCG) and d(CDCGTG) which contain thymine/2-aminoadenine (TD) base pairs. The latter structure has been solved and refined to 1.3 A resolution and it shows only small conformational changes due to the introduction of the TD base pairs in comparison with the structure of d(CG)3. Spectroscopic studies with these compounds demonstrate that DNA molecules containing 2-aminoadenine residues form Z-DNA slightly more easily than do those containing adenine nucleotides, but not as readily as the parent sequence containing only guanine-cytosine base pairs.     

Determination of the base composition of deoxyribonucleic acid by measurement of the adenine/guanine ratio

A method is described for determination of the base composition (as guanine+cytosine or adenine+thymine content) of DNA by accurate measurement of the adenine/guanine ratio. The DNA is hydrolysed with 0.03n-hydrochloric acid for 40min. to release the purines. The hydrolysate is subjected to ion-exchange chromatography on Zeo-Karb 225. Apurinic acids are eluted with 0.03n-hydrochloric acid and then guanine and adenine are eluted separately with 2n-hydrochloric acid. Guanine and adenine are each collected as a single fraction, and the amount of base in each case is determined by measuring the volume and the extinction at suitable wavelengths. For use in the calculations, millimolar extinction coefficients in 2n-hydrochloric acid of 12.09 for adenine at 262mmu, and 10.77 for guanine at 248mmu, were determined with authentic samples of bases. The method gives extremely reproducible results: from 12 determinations with calf thymus DNA the adenine/guanine molar ratio had a standard deviation of 0.011; this corresponds to a standard deviation in guanine+cytosine content of 0.2% guanine+cytosine.