A Role for the Mutagenic DNA Self-Catalyzed Depurination Mechanism in the Evolution of 7SL-Derived RNAs.

The Alu element, the most prevalent SINE (short interspersed element) in the human genome, is one of the many RNA-encoding genes that evolved from the 7SL RNA gene. During analysis of the evolution of 7SL-derived RNAs, two distinct evolutionary intermediates capable of self-catalyzed DNA depurination (SDP) were identified. These SDP sequences spontaneously create apurinic sites that can result in increased mutagenesis due to their error-prone repair. This DNA self-depurination mechanism has been shown both in vitro and in vivo to lead to substitution and short frameshift mutations at a frequency that far exceeds their occurrence due to random errors in DNA replication. In both evolutionary intermediates, the same self-depurination sequence overlaps motifs necessary for successful transcription and SRP9/14 (signal recognition particle) binding; hence, mutations in this region could disrupt RNA activity. Yet, the 7SL-derived RNAs that arose from the elements capable of SDP show significant diversity in this region, and every new sequence retains the transcription and SRP9/14-binding motifs, even as it has lost the SDP sequence. While some (but not all) of the mutagenesis can be alternatively attributed to CpG decay, the very fact that the self-depurinating sequences are selectively discarded in all cases suggests that this was evolutionarily motivated to prevent further destructive mutagenesis by the SDP mechanism.

Site-Specific Self-Catalyzed DNA Depurination: A Biological Mechanism That Leads to Mutations and Creates Sequence Diversity.

Self-catalyzed DNA depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the 5'G residue of the 5'GA/TGG loop and of the first 5'A residue of the 5'GAGA loop, together with particular first stem base pairs, specifies their hydrolysis without involving protein, cofactor, or cation. As such, this mechanism is the only known DNA catalytic activity exploited by nature. The consensus sequences for self-depurination of such G- and A-loop residues occur in all genomes examined across the phyla, averaging one site every 2,000-4,000 base pairs. Because apurinic sites are subject to error-prone repair, leading to substitution and short frameshift mutations, they are both a source of genome damage and a means for creating sequence diversity. Their marked overrepresentation in genomes, and largely unchanging density from the lowest to the highest organisms, indicate their selection over the course of evolution. The mutagenicity at such sites in many human genes is associated with loss of function of key proteins responsible for diverse diseases.

Why the DNA self-depurination mechanism operates in HB-ß but not in ß-globin paralogs HB-δ, HB-ɛ1, HB-γ1 and HB-γ2.

The human ß-globin, δ-globin and ɛ-globin genes contain almost identical coding strand sequences centered about codon 6 having potential to form a stem-loop with a 5'GAGG loop. Provided with a sufficiently stable stem, such a structure can self-catalyze depurination of the loop 5'G residue, leading to a potential mutation hotspot. Previously, we showed that such a hotspot exists about codon 6 of ß-globin, with by far the highest incidence of mutations across the gene, including those responsible for 6 anemias (notably Sickle Cell Anemia) and ß-thalassemias. In contrast, we show here that despite identical loop sequences, there is no mutational hotspot in the δ- or ɛ1-globin potential self-depurination sites, which differ by only one or two base pairs in the stem region from that of the ß-globin gene. These differences result in either one or two additional mismatches in the potential 7-base pair-forming stem region, thereby weakening its stability, so that either DNA cruciform extrusion from the duplex is rendered ineffective or the lifetime of the stem-loop becomes too short to permit self-catalysis to occur. Having that same loop sequence, paralogs HB-γ1 and HB-γ2 totally lack stem-forming potential. Hence the absence in δ- and ɛ1-globin genes of a mutational hotspot in what must now be viewed as non-functional homologs of the self-depurination site in ß-globin. Such stem-destabilizing variants appeared early among vertebrates and remained conserved among mammals and primates. Thus, this study has revealed conserved sequence determinants of self-catalytic DNA depurination associated with variability of mutation incidence among human ß-globin paralogs.

Self-catalytic DNA depurination underlies human ß-globin gene mutations at codon 6 that cause anemias and thalassemias.

The human ß-globin gene contains an 18-nucleotide coding strand sequence centered at codon 6 and capable of forming a stem-loop structure that can self-catalyze depurination of the 5'G residue of that codon. The resultant apurinic lesion is subject to error-prone repair, consistent with the occurrence about this codon of mutations responsible for 6 anemias and ß-thalassemias and additional substitutions without clinical consequences. The 4-residue loop of this stem-loop-forming sequence shows the highest incidence of mutation across the gene. The loop and first stem base pair-forming residues appeared early in the mammalian clade. The other stem-forming segments evolved more recently among primates, thereby conferring self-depurination capacity at codon 6. These observations indicate a conserved molecular mechanism leading to ß-globin variants underlying phenotypic diversity and disease.

Self-catalyzed site-specific depurination of G residues mediated by cruciform extrusion in closed circular DNA plasmids.

A major variety of "spontaneous" genomic damage is endogenous generation of apurinic sites. Depurination rates vary widely across genomes, occurring with higher frequency at "depurination hot spots." Recently, we discovered a site-specific self-catalyzed depurinating activity in short (14-18 nucleotides) DNA stem-loop-forming sequences with a 5'-G(T/A)GG-3' loop and T·A or G·C as the first base pair at the base of the loop; the 5'-G residue of the loop self-depurinates at least 10(5)-fold faster than random "spontaneous" depurination at pH 5. Formation of the catalytic intermediate for self-depurination in double-stranded DNA requires a stem-loop to extrude as part of a cruciform. In this study, evidence is presented for self-catalyzed depurination mediated by cruciform formation in plasmid DNA in vitro. Cruciform extrusion was confirmed, and its extent was quantitated by digestion of the plasmid with single strand-specific mung bean endonuclease, followed by restriction digestion and sequencing of resulting mung bean-generated fragments. Appearance of the apurinic site in the self-depurinating stem-loop was confirmed by digestion of plasmid DNA with apurinic endonuclease IV, followed by primer extension and/or PCR amplification to detect the endonuclease-generated strand break and identify its location. Self-catalyzed depurination was contingent on the plasmid being supercoiled and was not observed in linearized plasmids, consistent with the presence of the extruded cruciform in the supercoiled plasmid and not in the linear one. These results indicate that self-catalyzed depurination is not unique to single-stranded DNA; rather, it can occur in stem-loop structures extruding from double-stranded DNA and therefore could, in principle, occur in vivo.

The consensus sequence for self-catalyzed site-specific G residue depurination in DNA.

The sequence variation tolerated within the stem-loop-forming genomic consensus sequence for self-catalyzed site-specific depurination of G residues is explored. The variation in self-depurination kinetics with sequence changes in the loop residues and stem base pairs, as well as with pH, provides insights into the self-catalytic mechanism. The observations suggest that self-catalyzed depurination of the 5' G residue of the loop consensus sequence 5'-G(T/A)GG-3' probably involves formation of some intraloop hydrogen-bonded base pair with the 3'-terminal G residue; although the electronic structure of both these G residues is retained, their 2-amino substituents are not critical for that interaction. The strong dependence of the self-depurination kinetics on stem stability suggests that the lifetime of some strained form of the loop is controlled by the integrity of the stem. In addition to the effects of length and base pair sequence on stem stability, there is a base pair requirement at the base of the loop: self-depurination is suppressed by 5'-C·G-3', 5'-A·T-3', or a mismatch but is most favored by 5'T·A3' and less so by 5'-G·C-3'. The occurrence in T and G of a similarly located carbonyl capable of hydrogen-bonding to the water molecule required for glycosyl bond hydrolysis may explain this sequence requirement. In toto, the more complete definition of the consensus sequence provided by this investigation enables a more accurate estimation of their number in the human genome and their distribution among different genes.

Self-catalyzed site-specific depurination of guanine residues within gene sequences.

A self-catalyzed, site-specific guanine-depurination activity has been found to occur in short gene sequences with a potential to form a stem-loop structure. The critical features of that catalytic intermediate are a 5'-G-T-G-G-3' loop and an adjacent 5'-T.A-3' base pair of a short duplex stem stable enough to fix the loop structure required for depurination of its 5'-G residue. That residue is uniquely depurinated with a rate some 5 orders of magnitude faster than that of random "spontaneous" depurination. In contrast, all other purine residues in the sequence depurinate at the spontaneous background rate. The reaction requires no divalent cations or other cofactors and occurs under essentially physiological conditions. Such stem-loops can form in duplex DNA under superhelical stress, and their critical sequence features have been found at numerous sites in the human genome. Self-catalyzed stem-loop-mediated depurination leading to flexible apurinic sites may therefore serve some important biological role, e.g., in nucleosome positioning, genetic recombination, or chromosome superfolding.

Are stop codons recognized by base triplets in the large ribosomal RNA subunit?

The precise mechanism of stop codon recognition in translation termination is still unclear. A previously published study by Ivanov and colleagues proposed a new model for stop codon recognition in which 3-nucleotide Ter-anticodons within the loops of hairpin helices 69 (domain IV) and 89 (domain V) in large ribosomal subunit (LSU) rRNA recognize stop codons to terminate protein translation in eubacteria and certain organelles. We evaluated this model by extensive bioinformatic analysis of stop codons and their putative corresponding Ter-anticodons across a much wider range of species, and found many cases for which it cannot explain the stop codon usage without requiring the involvement of one or more of the eight possible noncomplementary base pairs. Involvement of such base pairs may not be structurally or thermodynamically damaging to the model. However, if, according to the model, Ter-anticodon interaction with stop codons occurs within the ribosomal A-site, the structural stringency which that site imposes on sense codon.tRNA anticodon interaction should also extend to stop codon.Ter-anticodon interactions. Moreover, with Ter-tRNA in place of an aminoacyl-tRNA, for each of the various Ter-anticodons there is a sense codon that can interact with it preferentially by complementary and wobble base-pairing. Both these considerations considerably weaken the arguments put forth previously.

Stabilization of nucleic acid triplexes by high concentrations of sodium and ammonium salts follows the Hofmeister series.

The thermal stability of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6) and d(T)(21):d(A)(21);d(T)(21) was studied in the presence of high concentrations of the anions Cl(-), HPO(4)(2-), CH(3)COO(-), SO(4)(2-) and ClO(4)(-). Thermally-induced triplex and duplex transitions were identified by UV- and CD-spectroscopy and T(m) values were determined from melting profiles. A thermodynamic analysis of triplex transitions shows the limitations of commonly used treatments for determining the associated release or uptake of salt, solute or water. Enhancement of the stability of these triplexes follows the rank order of the Hofmeister series for anions of sodium and ammonium salts, whereas water structure-breaking solutes have the opposite effect. The rank order for the Hofmeister series ClO(4)(-)

Enhanced stabilization of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6), d(T)(21):d(A)(21);d(T)(21) and poly r(U:AU) by water structure-making solutes.

A variety of organic cations, cationic lipids, low molecular weight alcohols, sodium dodecylsulfate, trehalose, glycerol, low molecular weight polyethylene glycols, and DMSO were tested for their ability to modulate the stability of the triplexes d(C(+)-T)(6):d(A-G)(6);d(C-T)(6), d(T)(21):d(A)(21);d(T)(21), poly r(U:A U) and their respective core duplexes, d(A-G)(6);d(C-T)(6), d(A)(21);d(T)(21), poly r(A-U). Very substantial enhancement of triplex stability over that in a physiological salt buffer at pH 7 is obtained with different combinations of triplex and high concentrations of these additives, e.g. trimethylammonium chloride and d(C(+)-T)(6):d(A-G)(6);d(C-T)(6); 2-propanol and d(T)(21):d(A)(21);d(T)(21); ethanol and poly r(U:A;U). Triplex formation is even observed with a 1:1 strand mixture of d(A-G)(6) and d(C-T)(6) in the presence of dimethylammonium, tetramethylammonium, and tetraethylammonium-chloride, as well as methanol, ethanol, and 2-propanol. Triplex stability follows the water structure-making ability (and in some cases the duplex unwinding ability) of the organic cations, the low molecular weight alcohols and other neutral organic compounds, whereas water structure-breaking additives decrease triplex stability. These findings are consistent with those reported in the accompanying paper that triplex formation occurs with a net uptake of water. Since the findings suggest that third strand-binding is facilitated by unwinding of the target duplex, it is inferred that triplex formation may be enhanced by nucleic acid binding proteins operating similarly.

Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence.

Three identical deoxyoligonucleotide third strands with a 3'-terminal psoralen moiety attached by linkers that differ in length (N = 16, 6 and 4 atoms) and structure were examined for their ability to form triplex-directed psoralen photoproducts with both the mutant T residue of the Sickle Cell beta-globin gene and the comparable wild-type sequence in linear duplex targets. Specificity and yield of UVA (365 nm) and visible (419 nm) light-induced photoadducts were studied. The total photoproduct yield varies with the linker and includes both monoadducts and crosslinks at various available pyrimidine sites. The specificity of photoadduct formation at the desired mutant T residue site was greatly improved by shortening the psoralen linker. In particular, using the N-4 linker, psoralen interaction with the residues of the non-coding duplex strand was essentially eliminated, while modification of the Sickle Cell mutant T residue was maximized. At the same time, the proportion of crosslink formation at the mutant T residue upon UV irradiation was much greater for the N-4 linker. The photoproducts formed with the wild-type target were fully consistent with its single base pair difference. The third strand with the N-4 linker was also shown to bind to a supercoiled plasmid containing the Sickle Cell mutation site, giving photoproduct yields comparable with those observed in the linear mutant target.

Repairing the Sickle Cell mutation. III. Effect of irradiation wavelength on the specificity and type of photoproduct formed by a 3'-terminal psoralen on a third strand directed to the mutant base pair.

Using a psoralen delivery system mediated by a DNA third strand that binds selectively to linear target duplexes immediately downstream from the Sickle Cell beta-globin gene mutation and the comparable wild-type beta-globin gene sequence, the kinetics of formation and yield of psoralen monoadducts and crosslinks with pyrimidine residues at and near the mutant base pair site and its wild-type counterpart were determined. By exploiting irradiation specificities at 300, 365 and 419 nm, it was possible to evaluate the orientation equilibrium of 3'-linked intercalated psoralen and to develop conditions that lead to preferential formation of each type of photoproduct in both the mutant and wild-type sequences. This makes possible the preparation of each type of photoproduct for use as a substrate for DNA repair. In this way, the base pair change(s) that each generates can be established.

Greater GNN pattern bias in sequence elements encoding conserved residues of ancient proteins may be an indicator of amino acid composition of early proteins.

The possibility that RNY pattern bias in extant sequences is a remnant of more pronounced bias of this type in early ancestors was investigated. To this end, conserved residues (those residues for which the inferred ancestral and known descendant amino acids are identical) and non-conserved residues of ancient proteins dating to the Last Universal Ancestor were identified within six species: two archaea, two eubacteria and two eukaryotes. Bias within sequence elements encoding each subset of residues, conserved and non-conserved, was then determined. In all species, GNN bias is greater within conserved than non-conserved sequence elements, whereas ANN is not. This difference is statistically significant in all six species examined. Since the relative mutability of the GNN-encoded amino acids does not explain the greater bias in conserved sequences, it is concluded that early sequences probably possessed a strong GNN bias. It is suggested that this bias may be a consequence of the GNN codons being the first introduced into the genetic code. Although NNY bias is also greater within conserved sequence elements of the six species, that difference is statistically significant in only half of them. Therefore, the evidence for early NNY bias remains inconclusive. The findings of this study do not support the proposal of Diaz-Lazcoz et al. (J. Mol. Biol. 250 (1995) 123) that the codons of the TCN four-codon block were the first assigned to serine during the evolution of the genetic code.

Evolution of amino acid frequencies in proteins over deep time: inferred order of introduction of amino acids into the genetic code.

To understand more fully how amino acid composition of proteins has changed over the course of evolution, a method has been developed for estimating the composition of proteins in an ancestral genome. Estimates are based upon the composition of conserved residues in descendant sequences and empirical knowledge of the relative probability of conservation of various amino acids. Simulations are used to model and correct for errors in the estimates. The method was used to infer the amino acid composition of a large protein set in the Last Universal Ancestor (LUA) of all extant species. Relative to the modern protein set, LUA proteins were found to be generally richer in those amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those amino acids that are believed to have been unavailable or scarce. It is proposed that the inferred amino acid composition of proteins in the LUA probably reflects historical events in the establishment of the genetic code.

Increased frequency of cysteine, tyrosine, and phenylalanine residues since the last universal ancestor.

Analysis of extant proteomes has the potential of revealing how amino acid frequencies within proteins have evolved over biological time. Evidence is presented here that cysteine, tyrosine, and phenylalanine residues have substantially increased in frequency since the three primary lineages diverged more than three billion years ago. This inference was derived from a comparison of amino acid frequencies within conserved and non-conserved residues of a set of proteins dating to the last universal ancestor in the face of empirical knowledge of the relative mutability of these amino acids. The under-representation of these amino acids within last universal ancestor proteins relative to their modern descendants suggests their late introduction into the genetic code. Thus, it appears that extant ancient proteins contain evidence pertaining to early events in the formation of biological systems.