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.

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.

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.

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-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.