NMR scalar couplings across Watson–Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy

This paper describes the NMR observation of 15N—15N and 1H—15N scalar couplings across the hydrogen bonds in Watson–Crick base pairs in a DNA duplex, hJNN and hJHN. These couplings represent new parameters of interest for both structural studies of DNA and theoretical investigations into the nature of the hydrogen bonds. Two dimensional [15N,1H]-transverse relaxation-optimized spectroscopy (TROSY) with a 15N-labeled 14-mer DNA duplex was used to measure hJNN, which is in the range 6–7 Hz, and the two-dimensional hJNN-correlation-[15N,1H]-TROSY experiment was used to correlate the chemical shifts of pairs of hydrogen bond-related 15N spins and to observe, for the first time, hJHN scalar couplings, with values in the range 2–3.6 Hz. TROSY-based studies of scalar couplings across hydrogen bonds should be applicable for large molecular sizes, including protein-bound nucleic acids.

Nucleic acid duplexes incorporating a dissociable covalent base pair

We have used molecular modeling techniques to design a dissociable covalently bonded base pair that can replace a Watson-Crick base pair in a nucleic acid with minimal distortion of the structure of the double helix. We introduced this base pair into a potential precursor of a nucleic acid double helix by chemical synthesis and have demonstrated efficient nonenzymatic template-directed ligation of the free hydroxyl groups of the base pair with appropriate short oligonucleotides. The nonenzymatic ligation reactions, which are characteristic of base paired nucleic acid structures, are abolished when the covalent base pair is reduced and becomes noncoplanar. This suggests that the covalent base pair linking the two strands in the duplex is compatible with a minimally distorted nucleic acid double-helical structure.

The role of mismatch repair in the prevention of base pair mutations in Saccharomyces cerevisiae

In most organisms, the mismatch repair (MMR) system plays an important role in substantially lowering mutation rates and blocking recombination between nonidentical sequences. In Saccharomyces cerevisiae, the products of three genes homologous to Escherichia coli mutS—MSH2, MSH3, and MSH6—function in MMR by recognizing mispaired bases. To determine the effect of MMR on single-base pair mismatches, we have measured reversion rates of specific point mutations in the CYC1 gene in both wild-type and MMR-deficient strains. The reversion rates of all of the point mutations are similar in wild-type cells. However, we find that in the absence of MSH2 or MSH6, but not MSH3, reversion rates of some mutations are increased by up to 60,000-fold, whereas reversion rates of other mutations are essentially unchanged. When cells are grown anaerobically, the reversion rates in MMR-deficient strains are decreased by as much as a factor of 60. We suggest that the high reversion rates observed in these MMR-deficient strains are caused by misincorporations opposite oxidatively damaged bases and that MMR normally prevents these mutations. We further suggest that recognition of mispairs opposite damaged bases may be a more important role for MMR in yeast than correction of errors opposite normal bases.

Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicals.

A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.

Evidence for DNA phosphate backbone alkylation and cleavage by pyrrolo[1,2-a]benzimidazoles: small molecules capable of causing base-pair-specific phosphodiester bond hydrolysis.

This report presents evidence that a reduced pyrrolo[1,2-a]benzimidazole (PBI) cleaves DNA as a result of phosphate alkylation followed by hydrolysis of the resulting phosphate triester. The base-pair specificity of the phosphate alkylation results from Hoogsteen-type hydrogen bonding of the reduced PBI in the major groove at only A.T and G.C base pairs. Alkylated phosphates were detected by 31P NMR and the cleavage products were detected by 1H NMR and HPLC. Evidence is also presented that a reduced PBI interacts with DNA in the major groove rather than in the minor groove or by intercalation.

The crystal structure of the Rev binding element of HIV-1 reveals novel base pairing and conformational variability

The crystal and molecular structure of an RNA duplex corresponding to the high affinity Rev protein binding element (RBE) has been determined at 2.1-Å resolution. Four unique duplexes are present in the crystal, comprising two structural variants. In each duplex, the RNA double helix consists of an annealed 12-mer and 14-mer that form an asymmetric internal loop consisting of G-G and G-A noncanonical base pairs and a flipped-out uridine. The 12-mer strand has an A-form conformation, whereas the 14-mer strand is distorted to accommodate the bulges and noncanonical base pairing. In contrast to the NMR model of the unbound RBE, an asymmetric G-G pair with N2-N7 and N1-O6 hydrogen bonding, is formed in each helix. The G-A base pairing agrees with the NMR structure in one structural variant, but forms a novel water-mediated pair in the other. A backbone flip and reorientation of the G-G base pair is required to assume the RBE conformation present in the NMR model of the complex between the RBE and the Rev peptide.

Recognition of nonhybridizing base pairs during nucleotide excision repair of DNA

Nondistorting C4′ backbone adducts serve as molecular tools to analyze the strategy by which a limited number of human nucleotide excision repair (NER) factors recognize an infinite variety of DNA lesions. We have constructed composite DNA substrates containing a noncomplementary site adjacent to a nondistorting C4′ adduct to show that the loss of hydrogen bonding contacts between partner strands is an essential signal for the recruitment of NER enzymes. This specific conformational requirement for excision is mediated by the affinity of xeroderma pigmentosum group A (XPA) protein for nonhybridizing sites in duplex DNA. XPA recognizes defective Watson–Crick base pair conformations even in the absence of DNA adducts or other covalent modifications, apparently through detection of hydrophobic base components that are abnormally exposed to the double helical surface. This recognition function of XPA is enhanced by replication protein A (RPA) such that, in combination, XPA and RPA constitute a potent molecular sensor of denatured base pairs. Our results indicate that the XPA–RPA complex may promote damage recognition by monitoring Watson–Crick base pair integrity, thereby recruiting the human NER system preferentially to sites where hybridization between complementary strands is weakened or entirely disrupted.

Unnatural base pairs for specific transcription

An unnatural base pair of 2-amino-6-(N,N-dimethylamino)purine (designated as x) and pyridin-2-one (designated as y) has been developed for specific transcription. The ribonucleoside triphosphates of y and a modified y, 5-methylpyridin-2-one, are selectively incorporated into RNA opposite x in the templates by T7 RNA polymerase. In addition, the sequences of the DNA templates containing x can be confirmed by a dideoxynucleotide chain-terminator method supplemented with the deoxynucleoside triphosphate of y. The bulky dimethylamino group of x in the templates effectively eliminates noncognate pairing with the natural bases. These results enable RNA biosynthesis for the specific incorporation of unnatural nucleotides at the desired positions.

Architectural specificity in chromatin structure at the TATA box in vivo: Nucleosome displacement upon β-phaseolin gene activation

Extensive studies of the β-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2

Mutations are introduced into rearranged Ig variable genes at a frequency of 10−2 mutations per base pair by an unknown mechanism. Assuming that DNA repair pathways generate or remove mutations, the frequency and pattern of mutation will be different in variable genes from mice defective in repair. Therefore, hypermutation was studied in mice deficient for either the DNA nucleotide excision repair gene Xpa or the mismatch repair gene Pms2. High levels of mutation were found in variable genes from XPA-deficient and PMS2-deficient mice, indicating that neither nucleotide excision repair nor mismatch repair pathways generate hypermutation. However, variable genes from PMS2-deficient mice had significantly more adjacent base substitutions than genes from wild-type or XPA-deficient mice. By using a biochemical assay, we confirmed that tandem mispairs were repaired by wild-type cells but not by Pms2−/− human or murine cells. The data indicate that tandem substitutions are produced by the hypermutation mechanism and then processed by a PMS2-dependent pathway.

Activation of microhelix charging by localized helix destabilization

We report that aminoacylation of minimal RNA helical substrates is enhanced by mismatched or unpaired nucleotides at the first position in the helix. Previously, we demonstrated that the class I methionyl-tRNA synthetase aminoacylates RNA microhelices based on the acceptor stem of initiator and elongator tRNAs with greatly reduced efficiency relative to full-length tRNA substrates. The cocrystal structure of the class I glutaminyl-tRNA synthetase with tRNAGln revealed an uncoupling of the first (1⋅72) base pair of tRNAGln, and tRNAMet was proposed by others to have a similar base-pair uncoupling when bound to methionyl-tRNA synthetase. Because the anticodon is important for efficient charging of methionine tRNA, we thought that 1⋅72 distortion is probably effected by the synthetase–anticodon interaction. Small RNA substrates (minihelices, microhelices, and duplexes) are devoid of the anticodon triplet and may, therefore, be inefficiently aminoacylated because of a lack of anticodon-triggered acceptor stem distortion. To test this hypothesis, we constructed microhelices that vary in their ability to form a 1⋅72 base pair. The results of kinetic assays show that microhelix aminoacylation is activated by destabilization of this terminal base pair. The largest effect is seen when one of the two nucleotides of the pair is completely deleted. Activation of aminoacylation is also seen with the analogous deletion in a minihelix substrate for the closely related isoleucine enzyme. Thus, for at least the methionine and isoleucine systems, a built-in helix destabilization compensates in part for the lack of presumptive anticodon-induced acceptor stem distortion.

The U5 RNA of trypanosomes deviates from the canonical U5 RNA: The Leptomonas collosoma U5 RNA and its coding gene

Fractionation of the abundant small ribonucleoproteins (RNPs) of the trypanosomatid Leptomonas collosoma revealed the existence of a group of unidentified small RNPs that were shown to fractionate differently than the well-characterized trans-spliceosomal RNPs. One of these RNAs, an 80-nt RNA, did not possess a trimethylguanosine (TMG) cap structure but did possess a 5′ phosphate terminus and an invariant consensus U5 snRNA loop 1. The gene coding for the RNA was cloned, and the coding region showed 55% sequence identity to the recently described U5 homologue of Trypanosoma brucei [Dungan, J. D., Watkins, K. P. & Agabian, N. (1996) EMBO J. 15, 4016–4029]. The L. collosoma U5 homologue exists in multiple forms of RNP complexes, a 10S monoparticle, and two subgroups of 18S particles that either contain or lack the U4 and U6 small nuclear RNAs, suggesting the existence of a U4/U6⋅U5 tri-small nuclear RNP complex. In contrast to T. brucei U5 RNA (62 nt), the L. collosoma homologue is longer (80 nt) and possesses a second stem–loop. Like the trypanosome U3, U6, and 7SL RNA genes, a tRNA gene coding for tRNACys was found 98 nt upstream to the U5 gene. A potential for base pair interaction between U5 and SL RNA in the 5′ splice site region (positions −1 and +1) and downstream from it is proposed. The presence of a U5-like RNA in trypanosomes suggests that the most essential small nuclear RNPs are ubiquitous for both cis- and trans-splicing, yet even among the trypanosomatids the U5 RNA is highly divergent.

Use of intrinsic binding energy for catalysis by an RNA enzyme

The contribution of several individual ribozyme⋅substrate base pairs to binding and catalysis has been investigated using hammerhead ribozyme substrates that were truncated at their 3′ or 5′ ends. The base pairs at positions 1.1–2.1 and 15.2–16.2, which flank the conserved core, each contribute 104-fold in the chemical step, without affecting substrate binding. In contrast, base pairs distal to the core contribute to substrate binding but have no effect on the chemical step. These results suggest a “fraying model” in which each ribozyme⋅substrate helix can exist in either an unpaired (“open”) state or a helical (“closed”) state, with the closed state required for catalysis. The base pairs directly adjacent to the conserved core contribute to catalysis by allowing the closed state to form. Once the number of base pairs is sufficient to ensure that the closed helical state predominates, additional residues provide stabilization of the helix, and therefore increase binding, but have no further effect on the chemical step. Remarkably, the >5 kcal/mol free energy contribution to catalysis from each of the internal base pairs is considerably greater than the free energy expected for formation of a base pair. It is suggested that this unusually large energetic contribution arises because free energy that is typically lost in constraining residues within a base pair is expressed in the transition state, where it is used for positioning. This extends the concept of “intrinsic binding energy” from protein to RNA enzymes, suggesting that intrinsic binding energy is a fundamental feature of biological catalysis.

Mutagenicity in Escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde

The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido[1,2-α]purin-10(3H)-one (M1G) was determined by site-specific approaches using M13 vectors replicated in Escherichia coli. M1G was placed at position 6256 in the (−)-strand of M13MB102 by ligating the oligodeoxynucleotide 5′-GGT(M1G)TCCG-3′ into a gapped-duplex derivative of the vector. Unmodified and M1G-modified genomes containing either a cytosine or thymine at position 6256 of the (+)-strand were transformed into repair-proficient and repair-deficient E. coli strains, and base pair substitutions were quantitated by hybridization analysis. Modified genomes containing a cytosine opposite M1G resulted in roughly equal numbers of M1G→A and M1G→T mutations with few M1G→C mutations. The total mutation frequency was ≈1%, which represents a 500-fold increase in mutations compared with unmodified M13MB102. Transformation of modified genomes containing a thymine opposite M1G allowed an estimate to be made of the ability of M1G to block replication. The (−)-strand was replicated >80% of the time in the unadducted genome but only 20% of the time when M1G was present. Correction of the mutation frequency for the strand bias of replication indicated that the actual frequency of mutations induced by M1G was 18%. Experiments using E. coli with different genetic backgrounds indicated that the SOS response enhances the mutagenicity of M1G and that M1G is a substrate for repair by the nucleotide excision repair complex. These studies indicate that M1G, which is present endogenously in DNA of healthy human beings, is a strong block to replication and an efficient premutagenic lesion.

Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase

Oligonucleotides that recapitulate the acceptor stems of tRNAs are substrates for aminoacylation by many tRNA synthetases in vitro, even though these substrates are missing the anticodon trinucleotides of the genetic code. In the case of tRNAAla a single acceptor stem G⋅U base pair at position 3·70 is essential, based on experiments where the wobble pair has been replaced by alternatives such as I⋅U, G⋅C, and A⋅U, among others. These experiments led to the conclusion that the minor-groove free 2-amino group (of guanosine) of the G⋅U wobble pair is essential for charging. Moreover, alanine-inserting tRNAs (amber suppressors) that replace G⋅U with mismatches such as G⋅A and C⋅A are partially active in vivo and can support growth of an Escherichia coli tRNAAla knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition. Herein we investigate the charging in vitro of oligonucleotide and full-length tRNA substrates that contain mismatches at the position of the G⋅U pair. Although most of these substrates have undetectable activity, G⋅A and C⋅A variants retain some activity, which is, nevertheless, reduced by at least 100-fold. Thus, the in vivo assays are much less sensitive to large changes in aminoacylation kinetic efficiency of 3·70 variants than is the in vitro assay system. Although these functional data do not clarify all of the details, it is now clear that specific atomic groups are substantially more important in determining kinetic efficiency than is a helical distortion. By implication, the activity of mutant tRNAs measured in the in vivo assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.