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

Abstract

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 -T⅐A-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.DNA self-catalysis ͉ guanine depurination ͉ stem-loop structure D epurination in DNA has been recognized as a spontaneous form of intracellular DNA damage that affects both G and A residues in an essentially random way (1). In mammalian cells, such damage has been estimated to occur with a k obs of 3 ϫ 10 Ϫ9 min Ϫ1 (2). A complex and elaborate cellular DNA repair machinery has evolved to repair apurinic sites (3, 4). It has long been known that some G residues are more prone to depurination than others (5, 6), and such depurination hot spots have been associated with elevated mutation rates (6, 7). Notwithstanding these insights, notions to account for DNA depurination that go beyond recognition that hydrolysis of the purine-deoxyribose glycosyl bond is acid-catalyzed have not been forthcoming.In the course of studies using a 29-residue single-stranded coding strand fragment encompassing the mutation site of the sickle cell ␤-globin gene (8-10), we found that this 29-nt oligomer as well as its shortened 18-nt segment self-catalyze site-specific depurination of a singular G residue adjacent to the mutation site. In this report, we identify the precise location of the depurination site, the reaction mechanism and its immediate products, the structure of the catalytic intermediate, and some of the sequence requirements and their tolerance for variation within the intermediate. We also note the wide distribution of such putative self-depurinating sequences in the human genome. Results Specific Backbone Cleavage Is the Result of Site-Specific Depurination.It is as a consequence of slow spontaneous backbone cleavage at apurinic sites that the self-catalyzed site-specific depurination we report here was discovered. At the outset, it was observed that this cleavage occurs whether or not 10 Ϫ3 M EDTA is present, indicating that divalent cations are not required for the cleavage. Incubation of the highly purified and homogeneous 29-nt ol...

Self-catalytic DNA Depurination Underlies Human β-Globin Gene Mutations at Codon 6 That Cause Anemias and Thalassemias

“…We therefore propose that many of the recurrent mutations at ␤-globin codon 6 arose as a result of error-prone repair at the apurinic site generated by the site-specific self-catalytic depurination mechanism that has been experimentally established (3)(4)(5)16). In this light, the increased vulnerability to mutation brought about by the mechanism may well have been coupled on several independent occasions to a selectable fitness advantage stemming from resistance to malaria.…”

“…Rather, the sickle cell mutation enhances it, increasing the kinetics of self-depurination approximately 50-fold (3,4). In effect, this means that intrinsic to the sickle cell mutant is the ability to either revert to wild type or else to some other variant that may carry the potential to similarly provide resistance to malaria.…”

“…The evolutionary persistence of a stem base pair mismatch as part of these features is intriguing, particularly because mutations that convert the mismatch to a complementary base pair would enhance the self-catalytic activity considerably (3,4). Both possible mutations that remove the mismatch from the stem of the catalytic intermediate, thereby stabilizing it, have been identified and are not connected to any clinical pathology.…”

“…The DNA self-depurination mechanism was discovered in synthetic 29-residue single-stranded sequences containing a segment of the coding strand of HBB surrounding the sickle cell mutation site (3). The analyses in the present report are based upon a careful evaluation of the substitution and short deletion mutations recorded for the codons of the HBB gene in the Hb Var (1) and HGMD (2) databases and of HBB variants elsewhere in the literature.…”

“…1A) (3,4). As a consequence of its self-complementarity, such a palindromic sequence can extrude as a cruciform (5) (Fig.…”