Self-Catalyzed Site-Specific Depurination of Guanine Residues Within Gene Sequences

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

Olga Amosova*, Richard Coulter, and Jacques R. Fresco*

Department of Molecular Biology, Princeton University, Princeton, NJ 08544

Edited by Jack W. Szostak, Massachusetts General Hospital, Boston, MA, and approved January 19, 2006 (received for review September 28, 2005)

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 su- perfolding.

͉ ͉ DNA self-catalysis guanine depurination stem-loop structure Fig. 1. Localization of the cleavage site. A 32P-labeled 8-nt oligomer complementary to the 3Ј end of D-1 was used as a primer in a Sanger sequencing epurination in DNA has been recognized as a spontaneous reaction. The appearance of several Ϸ9- to 10-nt cleavage products in the D-1 32 form of intracellular DNA damage that affects both G and template lane labeled at the 5Ј end with P after piperidine treatment is D ␤ A residues in an essentially random way (1). In mammalian cells, consistent with site-specific depurination followed by -elimination. Ϫ9 such damage has been estimated to occur with a kobs of 3 ϫ 10 Ϫ1 min (2). A complex and elaborate cellular DNA repair ma- (11) was performed instead, only the Ϸ19-nt band was visible, chinery has evolved to repair apurinic sites (3, 4). It has long been whereas the shorter group of bands was barely visible (data not known that some G residues are more prone to depurination shown). The presence of the several bands refractory to terminal than others (5, 6), and such depurination hot spots have been deoxytransferase, with mobilities corresponding to Ϸ9–10 nt, is associated with elevated mutation rates (6, 7). Notwithstanding consistent with depurination that results in an equilibrium these insights, notions to account for DNA depurination that go between C1Ј hemiacetal and C1Ј aldehyde sugars at the apurinic beyond recognition that hydrolysis of the purine–deoxyribose site, accounting for two of the bands. Subsequent loss of either glycosyl bond is acid-catalyzed have not been forthcoming. In the course of studies using a 29-residue single-stranded the terminal phosphate only or both the phosphate and sugar, coding strand fragment encompassing the mutation site of the each resulting in successively increasing fragment mobility, sickle cell ␤-globin gene (8–10), we found that this 29-nt would account for the other two. Piperidine treatment at 75°C ␤ oligomer as well as its shortened 18-nt segment self-catalyze accelerates such backbone cleavage by base-catalyzed -elimi- site-specific depurination of a singular G residue adjacent to the nation (12, 13), thereby allowing detection and quantitation of mutation site. In this report, we identify the precise location of the accumulated apurinic sites. the depurination site, the reaction mechanism and its immediate The initial backbone cleavage site was located by comparing products, the structure of the catalytic intermediate, and some the large cleavage product with a sequencing ladder on dena- of the sequence requirements and their tolerance for variation turing PAGE (Fig. 1). The large cleavage product band corre- within the intermediate. We also note the wide distribution of sponded in mobility to a DNA polymerase product resulting such putative self-depurinating sequences in the human genome. from incorporation of 2Ј,3Ј-dideoxyadenosine opposite the sickle cell mutant T residue immediately downstream from the Results depurinated G. This band appeared equally intense in all four Specific Backbone Cleavage Is the Result of Site-Specific Depurination. residue lanes because of significant presence of the 19-nt cleav- It is as a consequence of slow spontaneous backbone cleavage at age product in the D-1 template, as revealed by polynucleotide apurinic sites that the self-catalyzed site-specific depurination we kinase labeling. The next band was also equally intense in the report here was discovered. At the outset, it was observed that this cleavage occurs whether or not 10Ϫ3 M EDTA is present, indicating that divalent cations are not required for the cleavage. Conflict of interest statement: No conflicts declared. Incubation of the highly purified and homogeneous 29-nt oli- This paper was submitted directly (Track II) to the PNAS office. gomer D-1 at 22°C (to allow depurination to proceed) sponta- Abbreviations: dG, deoxyguanosine; Exo III, exonuclease III. neously yielded a 19-nt fragment and an Ϸ9- to 10-nt group of *To whom correspondence may be addressed. E-mail: [email protected] or 32 four fragments revealed by labeling their 5Ј ends with PO4 (Fig. [email protected]. 1). However, when 3Ј-labeling with terminal deoxytransferase © 2006 by The National Academy of Sciences of the USA

4392–4397 ͉ PNAS ͉ March 21, 2006 ͉ vol. 103 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508499103 Downloaded by guest on September 24, 2021 Table 1. Some deoxyoligonucleotides used in the study Deoxyoligonucleotide Sequence

Anti-D-1 3Ј-GACTGAGGACACCTCTTCAGACGGCAATG-5Ј D-1 5Ј-CTGACTCCTGTGGAGAAGTCTGCCGTTAC-3Ј DH-1 5Ј-CTGACTCCTGAGGAGAAGTCTGCCGTTAC-3Ј D1-T 5Ј-CTGACTCCTTTGGAGAAGTCTGCCGTTAC-3Ј D1-A 5Ј-CTGACTCCTATGGAGAAGTCTGCCGTTAC-3Ј D1-C 5Ј-CTGACTCCTCTGGAGAAGTCTGCCGTTAC-3Ј

four lanes, consistent with the proclivity of DNA polymerase to Fig. 3. Piperidine cleavage of 29-nt sequence variants of the D-1 fragment of the sickle cell ␤-globin gene coding strand after 48-h incubation at 22°C (pH insert any residue opposite an apurinic site (14). Ј 5.0). No cleavage was evident when a pyrimidine residue replaced the specif- The location of the depurination site and the presence of a 5 ically depurinated G residue. Only a trace of cleavage product was evident phosphate on the 19-nt product of spontaneous cleavage were with an A substitution. confirmed by successful ligation of a synthetic 10-nt oligomer bearing a 3Ј-OH and a 5Ј-phosphate, and the gel-purified 19-nt spontaneous cleavage product after their annealing to the com- quence variants (Table 1) were compared. The site-specific plementary 29-nt anti-D-1 strand (see Table 1 and Fig. 2). depurination occured only when the residue 5Ј to the cleavage Ligation can succeed only when the two fragments annealed with site was G (Fig. 3). Its replacement by a pyrimidine eliminated the 29-nt complementary oligomer are immediately adjacent. cleavage, and only a trace of the cleavage product was visible for Because the synthetic 10-nt oligomer had a 3Ј-OH, the 19-nt the A replacement. Cleavage was significantly less for the fragment must bear a 5Ј-phosphate for ligation. In fact, ligation wild-type oligomer DH-1, indicating that the nature of the produced a full-size D-1 strand, as well as a D-1 dimer, a product immediate downstream residue is also important. It is worth of blunt-end ligation of two duplexes (Fig. 2, lane 1). Alkaline noting that all of these 29-nt oligomers had at least six other G phosphatase treatment of the 19-nt fragment before annealing residues in their sequences (seven in case of D-1 and DH-1, the resulted in only a trace of full-size D-1 (Fig. 2, lane 3). Because sickle cell and wild-type ␤-globin gene fragments, respectively); the integrity of the 19-nt fragment was unaffected by the yet none of them displayed any detectable depurination. Taken phosphatase treatment (data not shown), it must be the removal together, the results in Figs. 1 and 3 show that the depurination of its 5Ј-phosphate that prevents ligation. is very site-specific.

-A Guanine Residue Is Required 5؅ to the Cleavage Site. To determine Free Guanine Is One Initial Product of the Catalysis. With depurina the sequence requirements immediately adjacent to the cleavage tion presumed to be the primary event leading to backbone site that are essential for the depurination, several 29-nt se- cleavage, direct evidence was sought for free guanine and the resultant apurinic site. Fig. 4 compares HPLC profiles for incubated D-1, D1-T [which does not undergo piperidine- induced cleavage and therefore does not lose its base (Fig. 3)], and a mixture of D1-T and deoxyguanosine (dG) as a marker (neither dG nor guanine binds to the C18 column), with the molar ratio of dG:D1-T similar to that expected from the catalytic depurination of oligomer D-1. The small, immediately

Fig. 4. HPLC profiles of incubated D1-T, a mixture of D1-T͞dG, and incubated D-1 from a C18 reverse-phase column eluted with a 10–60% acetonitrile Fig. 2. Ligation of the 5Ј-32P-labeled 19-nt spontaneous cleavage product gradient (diagonal dotted line), started at 3 min, and ended at 13 min of and the 10-nt synthetic fragment on the complementary anti-D-1 template. elution in 25 mM sodium phosphate (pH 7.0). Strand concentrations were 0.05 Lane 1, ligation without prior phosphatase treatment. This denaturing gel mM, and free dG concentration was 0.01 mM. Neither dG nor guanine binds indicates that the unlabeled 19-nt fragment has a 5Ј-phosphate and so is to the column and is eluted within the first 4 min. The slight difference successfully ligated with the 10-nt oligomer on the complementary template. between the elution profiles of incubated D-1 and of the D1-T͞dG mixture is Lane 2, 32P-labeled 10-nt oligomer. Lane 3, ligation after alkaline phosphatase due to the deoxyribose moiety of the dG (deoxynucleoside) marker present in

treatment of the puriﬁed 19-nt fragment yields almost no ligated D-1. Lane 4, the ‘‘artiﬁcial’’ mixture and absent in the D-1 sample, because it is free guanine BIOCHEMISTRY 32P-labeled fresh D-1 stock. that is accumulated during the incubation.

Amosova et al. PNAS ͉ March 21, 2006 ͉ vol. 103 ͉ no. 12 ͉ 4393 Downloaded by guest on September 24, 2021 acid), this small peak displayed, in addition to UV absorption, strong blue fluorescence characteristic of free guanine. Thus, one primary product of self-catalytic depurination was defined.

Accumulation of Apurinic Sites Revealed by Piperidine Treatment Is Paralleled by Apurinic Endonuclease Cleavage. Fig. 5 illustrates accumulation of apurinic sites monitored both by piperidine cleavage (Fig. 5A) and by cleavage with an apurinic endonuclease (Fig. 5B). The apurinic endonuclease activity of exonuclease III (Exo III) was used for this purpose. When an 18-nt subfragment of the D-1 strand, D(-2), which is depurinated at the same rate as D-1 (Table 2), was incubated at pH 5 and 22°C for various times and then digested with Exo III, a 10-nt band, the equivalent of the 19-nt 3Ј-end digestion product of D-1, appeared on the gel. As in the case of piperidine cleavage, the digestion site was unique; but, unlike piperidine cleavage, which produces several short fragments, the Exo III 7-nt digestion product at the 5Ј side of the 18-nt D1(-2) was also unique (data not shown). The slope of the initial kinetics of cleavage was the same whether by piperidine or Exo III (Fig. 5C), indicating that they both reflect the rate of accumulation of apurinic sites. Hence, the self-catalyzed depurination gave rise to the apurinic sites that are cleavable by either piperidine treatment or apurinic endonuclease. It is also noteworthy that the kinetics of depurination, as measured by the yield of piperidine cleavage products, was independent of strand concentration over 2 orders of magnitude Ϫ4 Ϫ1 (Fig. 5D), with kobs ϭ 2 ϫ 10 min . This result is consistent with first-order kinetics and indicates that the catalytic intermediate is not a multistranded structure; rather, an intramolec- ular structure is critical for the self-catalytic depurination.

Self-Catalytic Depurination Correlates with Stability of a Stem-Loop Formed by D-1. The rate of catalytic depurination correlates with the ability of the oligomer to form a stem-loop with a 7-bp stem [including an A⅐Cϩ mismatch (15)] closed off at one end by a 4-nt loop, 5Ј-G-T-G-G-3Ј.Itisthe5Ј-G in this loop that undergoes the site-specific self-catalytic depurination. Table 2 indicates how the rate of this depurination changes with shortening of the oligonucleotide. Removal of 2 nt from the 5Ј end and 9 nt from the 3Ј end of the 29-nt D-1 gene fragment [D(-2)] had no effect on the depurination rate, apparently because it had no consequence for stem formation. However, further shortening reduced the number of possible base pairs in Fig. 5. Kinetics of apurinic site accumulation monitored by piperidine or the stem of the hairpin, obviously leading to its destabilization apurinic endonuclease cleavage. (A) Denaturing PAGE analysis of cleavage and, with that, reduction in the depurination rate as well. Thus, products of 3Ј-labeled D-1 samples incubated in 10 mM sodium cacodylate (pH when D-1 was shortened on either end, so that only 6 bp of the 5) at 22°C, with or without subsequent piperidine treatment at 75°C for 30 stem could form [D(-3) and D-19], the depurination rates fell min. Note that the cleavage products are highly homogeneous. (B) Exo III Ϸ5-fold; with only 5 bp possible, there was an Ϸ20-fold reduction digestion at the apurinic site produces a 10-nt cleavage product of the 18-nt (D-18). No depurination above the background rate was de- D-1 subfragment D(-2) labeled at the 3Ј end with terminal deoxytransferase. The band immediately below D(-2) is the n-1 oligomer that was not fully removed during PAGE purification. (C) Kinetics of self-catalyzed, site-specific Table 2. Reduction in the rate of self-catalytic depurination depurination revealed by Exo III digestion at apurinic sites (squares), piperi- when D-1 sequence is shortened dine cleavage (triangles), and nonspecific cleavage by piperidine (diamonds). Ϫ1 kobs for the depurination rate revealed by Exo III digestion coincides with that Oligomer Sequence kobs, min determined from piperidine-induced cleavage. kobs for the background- Ј Ј ϫ Ϫ4 nonspecific digestion rate is Ͻ10Ϫ6 minϪ1.(D) Kinetics of self-catalyzed site- D-1 5 -CTGACTCCTGTGGAGAAGTCTGCCGTTAC-3 2 10 Ϫ specific depurination of D-1 at three different concentrations over two orders D-20 5Ј-CTGACTCCTGTGGAGAAGTC-3Ј 2 ϫ 10 4 of magnitude. The apurinic sites were cleaved with piperidine at 70°C for 30 D(-2) 5Ј-GACTCCTGTGGAGAAGTC-3Ј 2 ϫ 10Ϫ4 min, and the yield of 19-nt cleavage product was determined after PAGE. D-19 5Ј-CTGACTCCTGTGGAGAAGT-3Ј 4 ϫ 10Ϫ5 Ϫ Ϫ Ϫ Diamonds, 5 ϫ 10 7 M D-1; squares, 5 ϫ 10 6 M D-1; triangles, 5 ϫ 10 5 M D-1; D(-3) 5Ј-ACTCCTGTGGAGAAGTC-3Ј 3.6 ϫ 10Ϫ5 filled circles, anti-D-1. D-18 5Ј-CTGACTCCTGTGGAGAAG-3Ј 1 ϫ 10Ϫ5 D-17 5Ј-CTGACTCCTGTGGAGAA-3Ј Ͻ10Ϫ6 D-14 5Ј-CTGACTCCTGTGGA-3Ј Ͻ10Ϫ6 eluted peak in the D1-T͞dG mixture was absent in the elution D(-6) 5Ј-CCTGTGGAGAAGTC-3Ј Ͻ10Ϫ6 profile of the D1-T sample alone, but such a peak was present in the elution profile of incubated D-1. When HPLC was The boldface G residue is depurinated, and residues that form stem base conducted under highly acidic conditions (10% trichloroacetic pairs are underlined.

4394 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508499103 Amosova et al. Downloaded by guest on September 24, 2021 Fig. 6. Temperature-dependence of site-specific and nonspecific spontaneous G residue depurination. Whereas spontaneous depurination progressively increases between 22°C and 65°C after 45 h of incubation, the temperature- Fig. 7. The stem-loop formed by the D-2 subfragment, and its digestion with dependence of the site-specific depurination is inverted. The reduction of mung bean endonuclease. (A) The minimal fully active self-depurinating specific G-residue depurination at higher temperatures reflects denaturation oligomer folds into a stem-loop structure with one A⅐Cϩ mismatch in the stem. of the catalytic intermediate stem-loop structure, such that the level of its The dotted arrow indicates the site of G-depurination, which is then cleaved depurination is the same as for the other G residues. by Exo III. The solid arrows indicate phosphodiester bonds digested by the single-strand-specific mung bean endonuclease. (B) PAGE analysis of mung bean endonuclease and Exo III digests (pH 5, 22°C) of the minimal fully active tected for D1(-6) or D-17, which have a potential to form only oligomer D1(-2) after labeling at the 3Ј end and a 24-h incubation at 22°C to 3 or 4 bp in the stem (one of them mismatched). Thus, the allow depurination to proceed. Lane 1, control (no added enzyme); lanes 2 and reduction in depurination rate correlated with the destabiliza- 3, mung bean nuclease digests after 1 and 5 min of incubation, respectively; tion of the hairpin structure. lane 4, Exo III digestion at the apurinic site. Formamide destabilizes base pair hydrogen bonds, reducing the T value of duplex DNA by Ϸ0.65°C for every 1% in the m tiates between the putative loop and stem residues, thereby solvent (16). Because the Tm value for the D-1 stem-loop is 42°C confirming the stem-loop structure. and a formamide concentration of 50% reduces Tm by Ϸ30°C, it is not surprising that the self-catalytic depurination activity is completely lost at 22°C. Effects of Sequence Variation Within the Stem-Loop Define Require- The effect of temperature on the depurination rate is also ments for the Self-Catalytic Depurination. Several 18-nt sequence Ј Ј informative. The rate of spontaneous acid-catalyzed depurina- variants of the stem closed by the 5 -G-T-G-G-3 loop were tion should follow the usual temperature dependence, doubling investigated: an ‘‘inverted’’ variant [D(-2)inv] in which the stem with every 10°C rise; the background depurination rate at 22°C strands were exchanged; a ‘‘perfect’’ variant [D(-2)p] in which Ϫ Ϫ ⅐ ϩ ⅐ of 3 ϫ 10 9 min 1 was determined by extrapolating rate data the A C mismatch was replaced by a G C base pair; a ‘‘changed’’ measured at 70°C based on this assumption. In contrast, the variant [D(-2)ch] with an arbitrary stem duplex sequence; and a site-specific self-catalytic depurination rate showed an inverse ‘‘disrupted’’ variant [D(-2)d] in which the number of potential dependence on temperature (Fig. 6). Thus, after 45 h of incu- mismatches in the stem should prevent duplex formation at room bation at 22°C, 70% of D-1 was cleaved at the specific depuri- temperature. As shown in Table 3, kobs for the perfect stem nation site by piperidine treatment, but only 40% was cleaved sequence was approximately twice that of the natural sickle cell ␤ after incubation at 43°C (just above Tm, where half the molecules -globin-derived sequence D(-2), suggesting that a more stable would be in the hairpin form), and only 16% was cleaved at 65°C. stem enhances the catalytic activity. The inverted variant showed This inverse dependence must reflect the thermal denaturation a substantial loss of activity even though a complementary of the stem-loop structure required for the self-catalysis. base-paired duplex structure was maintained. However, the The spontaneous depurination leading to nonspecific cleavage changed oligomer, in which only the first base pair adjacent to at other sites within the same molecule is also illustrated in Fig. the loop (5Ј-T⅐A-3Ј) and the A⅐Cϩ mismatch were conserved, 6. That process accelerated with increasing temperature, so that retained full activity. Thus, this T⅐A base pair appears to be depurination was the same (14–16%) for all of the G residues at essential for the self-catalytic activity. Moreover, when the stem 65°C. Taken together, these results are consistent with a stem- was fully complementary, just 5 bp were sufficient for full loop being the catalytic intermediate for the self-catalytic de- catalytic activity (D14p). Not surprisingly, the disrupted variant purination reaction.

The Stem-Loop Structure Is Confirmed by the Mung Bean Endonucle- Table 3. Effect of the stem-region sequence on the self-catalytic ase Digestion Pattern. A diagram of the putative stem-loop is depurination rate Ϫ1 shown along with the 18-nt subfragment cleavage pattern ob- Strand Sequence kobs, min tained with single-strand-specific mung bean endonuclease in Ϫ Fig. 7. Three major cutting sites are indicated in Fig. 7A, and D(-2)p 5Ј-GACTCCTGTGGAGGAGTC-3Ј 4 ϫ 10 4 Ϫ their fragmentation pattern is shown in Fig. 7B, lanes 2 and 3. D14p 5Ј-CTCCTGTGGAGGAG-3Ј 4 ϫ 10 4 Ј Ј ϫ Ϫ4 These cleavage sites surround the apurinic site localized by D(-2) 5 -GACTCCTGTGGAGAAGTC-3 2 10 Ј Ј ϫ Ϫ4 apurinic endonuclease (Fig. 7B, lane 4). The reason for much D(-2)ch 5 -GGACCCTGTGGAGAGTCG-3 2 10 Ј Ј ϫ Ϫ5 weaker digestion at the 3Ј end of the loop, where there are two D(-2)inv 5 -CTGAAGAGTGGTCCTCAG-3 3 10 Ј ЈϽϪ6 other G residues, may be their strong tendency to stack on each D(-2)d 5 -GACTCCTGTGGGAAGGTC-3 10

other and on the following 3Ј-base-paired A residue. Regardless, The boldface G residue is depurinated, and residues that form stem base BIOCHEMISTRY the mung bean endonuclease digestion pattern clearly differen- pairs are underlined.

Amosova et al. PNAS ͉ March 21, 2006 ͉ vol. 103 ͉ no. 12 ͉ 4395 Downloaded by guest on September 24, 2021 Discussion We have demonstrated a site-specific self-G-depurinating activity in a short DNA fragment of the sickle cell ␤-globin gene that is Ϸ3 ϫ 103 faster under physiological conditions (Ϸ105 faster at pH 5) than the spontaneous depurination rate (2). In this connection, the pH optimum shifts toward neutrality when the A⅐Cϩ base pair of the stem is replaced by a G⅐C base pair. Spontaneous depurination of DNA, a major type of endoge- nous DNA damage, is thought to be mediated by protonation of a G residue at N-7, with an intrinsic pKa of Ϸ2.3. Presumably, such protonation is also implicated in the self-catalyzed depurination, probably facilitated in part by intracellular macromolecular crowding known to alter acid-base equilibria (17). Because only one of the three G residues in the ‘‘catalytic loop’’ is depurinated, it is likely that some subtle aspect of the stem-loop structure contributes as well. We have shown that the self-catalytic depurination rate correlates with the ability of a single-stranded DNA fragment to form a stable hairpin with a 5Ј-G-T-G-G-3Ј loop and the adjacent 5Ј-T⅐A-3Ј base pair of the stem. The remaining sequence requirement is only for an additional number of complementary base pairs to stabilize the loop residues stacked on the T⅐A base pair. With increasing stability of the stem, helix breathing must be reduced, increasing the time average over which the catalytic conformation is maintained, hence a faster rate of depurination. Fig. 8. Self-catalytic site-specific G-depurination under physiological condi- The critical sequence features for this G-depurinating activity tions. (A) Denaturing PAGE analysis of piperidine cleavage products of 3Ј-32P- are present in numerous natural gene sequences. The activity is labeled D(-2)ch incubated in 20 mM Tris-HCl͞0.1 M NaCl͞0.05 M MgCl2 (pH 6.9) self-catalytic and requires no cofactors, and the consensus at 37°C. (B) Kinetics of self-catalyzed, site-specific depurination revealed by stem-loop structure is far smaller than the only reported site- piperidine cleavage at the specific site (diamonds) and nonspecific depurina- specific G-depurinating deoxyribozyme obtained by in vitro tion sites (circles). selection (18). The latter activity is contained within a complex three-stem-loop structure, requires divalent cations, and acts on a singular G residue in the three-residue loop of a stem-loop that exhibited no activity. These results define the required consensus is bound to the deoxyribozyme by complementary base pairing. sequence and structural elements. Thus, this catalyst depurinatesaGresidue of a deoxyoligomer substrate, acting like a true enzyme with turnover, unlike our Self-Catalytic Depurination Under Physiological Conditions. Al- activity, which does not represent catalysis in the enzymatic though the results presented above were obtained at pH 5 and sense because turnover is not possible. low salt concentration, the catalytic activity, albeit slightly Because the self-depurinating activity is associated with a reduced, was retained under essentially physiological conditions. single-stranded deoxyoligomer, it might be viewed as biologically Fig. 8 shows site-specific depurination kinetics revealed by irrelevant. However, superhelical stress in vivo induces formation piperidine cleavage of D(-2)ch after incubation at 37°C in 0.1 M of stem-loops in duplex DNA at inverted repeats (19, 20), and NaCl͞5 mM MgCl2͞20 mM Tris-HCl, pH 6.9͞20% PEG 1000 to stems as short as 5–7 bp easily extrude under physiological mimic intranuclear macromolecular crowding (17). Both the gel superhelical stress (21). and its quantitation clearly demonstrate the occurrence of the Many sites with a potential to form stem-loops are known to Ϫ6 Ϫ1 site-specific self-catalytic depurination; its kobs is 7 ϫ 10 min , be hypermutagenic (22, 23). The ability of some stem-loop Ϸ30 times slower than at pH 5, but still Ϸ3 ϫ 103 faster than structures to depurinate a specific loop residue could explain spontaneous background depurination. It is important to note such mutagenicity. But if the result of depurination were merely that for the D(-2)p strand, in which the A⅐Cϩ mismatch in the to mutilate or otherwise damage DNA, then we would expect stem is replaced with a G⅐C base pair, the reduction in the that evolution would have found a way to reduce the number of depurination rate under physiological conditions is only Ϸ15 such sites (by making use of alternative codons that would not times (data not shown), indicating that the acidic milieu required disturb encoded proteins but would disrupt stem base pairing). for maximum activity of the original sequence is due partially to Because this is clearly not the case, formation of apurinic sites the stabilization of the A⅐Cϩ mismatch. may serve some functional role. Thus, apurinic sites also slightly destabilize DNA duplexes (24) and increase local DNA flexi- bility (25), which may, for example, facilitate formation of open Potential Depurination Sites Are Abundant in the Human Genome. DNA–protein complexes and͞or play a role in DNA packaging Preliminary data mining was undertaken by using a Perl search in viruses, in nucleosome formation or positioning, or in facil- engine to find sequences in the human genome with a potential itating genetic recombination. to undergo self-catalytic G-depurination. We have already iden- With the discovery of a site-specific, self-catalytic, G- Ͼ tified 50,000 sites, distributed among all human chromosomes, depurinating activity intrinsic to numerous DNA sequences, we Ј Ј as having a central 5 -G-T-G-G-3 (loop) sequence, an adjacent are alerted to the possibility of yet other self-catalytic activities 5Ј-T⅐A-3Ј, and a capacity to form a stem of at least four contained within genomic DNA. additional complementary base pairs. Two of these sequences, both 14 nt in length, have been experimentally tested so far, and Materials and Methods each was found to possess self-depurinating activity with a kobs Deoxyoligonucleotides. Deoxyoligonucleotides were purchased of 5 ϫ 10Ϫ4 minϪ1 at pH 5. from Integrated DNA Technologies (Coralville, IA), purified to

4396 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508499103 Amosova et al. Downloaded by guest on September 24, 2021 homogeneity by denaturing PAGE, and recovered from gel slices and these data were used for the kinetic analysis. Each experi- by soaking overnight in 0.1 M sodium phosphate (pH 7.0). Final ment was performed at least three times, and the kinetic data purification was accomplished by 100% acetonitrile elution from were averaged. a C18 Sep-Pak reverse phase column (Waters) followed by spin evaporation. Oligomer concentration was then adjusted spec- Electrophoresis. Denaturing PAGE analysis and oligomer purifi- trophotometrically in Aldrich ACS reagent-grade water. cation were performed by using 16% slab gels 25 ϫ 45 cm in 8 M urea͞TBE at 1.5 kV for Ϸ 2h. 3؅-End Labeling. 3Ј-End labeling was performed by using terminal deoxytransferase (USB Corp.) (5 units) on Ϸ5 pmol of purified Sequencing. To determine the depurination͞cleavage position, oligomer with Ϸ5 pmol of [␣-32P]dideoxyATP (Amersham the oligomer was sequenced by using a Sequenase 2.0 kit (USB Pharmacia Biotech) (13). The labeled oligomers were purified by Corp.) and 32P-5Ј-end-labeled 8-nt primers complementary to denaturing PAGE before use. the 3Ј end of the 29-nt sickle cell ␤-globin gene fragment D-1.

-Kinetics of Depurination. Oligomers were incubated at 22°C in 10 Alkaline Phosphatase Treatment, Ligation, and 5؅-End Labeling. Stan mM sodium cacodylate (pH 5.0) (unless otherwise noted), and dard protocols were followed. aliquots were frozen on dry ice at appropriate intervals. To reveal apurinic sites, aliquots were treated with 0.1 M piperidine This paper is dedicated to Marianne Grunberg-Manago on the occasion for 30 min at 75°C to induce base-catalyzed backbone cleavage of her 85th birthday. We thank those who have graciously helped us with (12, 13). Either a trace amount of end-labeled oligomer was their expertise: Moshe Pritsker and Damian Glumcher with data mining, mixed with an unlabeled stock of appropriate concentration Nicole Ford with determining the concentration dependence of depurination kinetics, and Peter Wolanin with the HPLC analyses. We are also before incubation or the aliquots were labeled after the piper- grateful to Lucille Fresco-Cohen for a critical reading of the manuscript, idine treatment (both methods yield the same depurination to Ronald Breaker for valuable advice at the outset of this work, and to rates). Radioactivity of intact oligomer, its 3Ј-end cleavage Steven Broitman for directing R.C. to the project. This work was product, and nonspecific cleavage at other G residues were supported by National Institutes of Health Grants HL 63888 and CA quantitated by using a Molecular Dynamics PhosphorImager, 088547.

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