Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue in the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation.     

Cleavage of synthetic substrates containing non-nucleotide inserts by restriction endonucleases. Change in the cleavage specificity of endonuclease SsoII.

A study was made of the interaction between restriction endonucleases recognizing CCNGG (SsoII and ScrFI) or CCA/TGG (MvaI and EcoRII) DNA sequences and a set of synthetic substrates containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy] methylguanine (gIG) residues replacing either one of the central nucleosides or dG residues in the recognition site. The non-nucleotide inserts (except for gIG) introduced into the recognition site both increase the efficiency of SsoII and change its specificity. A cleavage at the noncanonical position takes place, in some cases in addition to the correct ones. Noncanonical hydrolysis by SsoII occurs at the phosphodiester bond adjacent to the point of modification towards the 5'-end. With the guanine base returned (the substrate with gIG), the correct cleavage position is restored. ScrFI specifically cleaves all the modified substrates. DNA duplexes with non-nucleotide inserts (except for the gIG-containing duplex) are resistant to hydrolysis by MvaI and EcoRII. Prompted by the data obtained we discuss the peculiarities of recognition by restriction endonucleases of 5-membered DNA sequences which have completely or partially degenerated central base pairs. It is suggested that SsoII forms a complex with DNA in an 'open' form.     

Properties of 2'-fluorothymidine-containing oligonucleotides: interaction with restriction endonuclease EcoRV

2'-Fluorothymidine (Tf) was synthesized via an improved procedure with (diethylamino)sulfur trifluoride. The compatibility of the analogue with DNA synthesis via the phosphoramidite method was demonstrated after complete enzymatic digestion of the oligonucleotides d(Tf11T) and d(Tf3T), the sole products of which were 2'-fluorothymidine and thymidine in the expected ratio. The 2'-fluorothymidine was also incorporated into the EcoRV recognition sequence (underlined), within the complementary oligonucleotides d(CAAACCGATATCGTTGTG) and d(CACAACGATATCGGTTTG). Thermal melting characteristics of these duplexes showed a significant decrease in stability only when both of the thymidine residues in one of the strands were replaced. In contrast, when all of one strand of a duplex contained 2'-fluorothymidine, as in d(Tf11T).d(A12), a substantially higher Tm and cooperativity of melting was observed relative to the unmodified structure. EcoRV cleaved a duplex that contained a 2'-fluorothymidine at the scissile linkage in each strand at two-thirds of the rate obtained for the unmodified structure. A duplex containing two 2'-fluorothymidine residues in one strand and none in the other was cleaved at one-third of the rate in its unsubstituted strand, whereas the cleavage rate was reduced to 22% in its modified strand.     

Change in the cleavage site of synthetic substrates of SsoII restriction endonucleases upon introduction of non-nucleotide inserts in the recognition segment]

The cleavage of synthetic DNA duplexes containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) residues instead of one of dG residues or one of the nucleosides of the central base pair of the recognition site by SsoII restriction endonuclease (decreases CCNGG) has been studied. It is found that the non-nucleotide insertions (except for glG) result in a change of the SsoII cleavage site and an increase of the efficiency of the cleavage. The novel noncanonical cleavage occurs at the phosphodiester bond adjoining the non-nucleotide insert from the 5'-end.     

The interaction of DNA duplexes containing 2-aminopurine with restriction endonucleases EcoRII and SsoII.

Oligonucleotides containing 2-aminopurine (2-AP) in place of G or A in the recognition site of EcoRII (CCT/AGG) or SsoII (CCNGG) restriction endonucleases have been synthesized in order to investigate the specific interaction of DNA with these enzymes. Physicochemical properties (CD spectra and melting behaviour) have shown that DNA duplexes containing 2-aminopurine exist largely in a stable B-like form. 2-Aminopurine base paired with cytidine, however, essentially influences the helix structure. The presence of a 2-AP-C mismatch strongly reduces the stability of the duplexes in comparison with the natural double strand, indicated by a biphasic melting behaviour. SsoII restriction endonuclease recognizes and cleaves the modified substrate with a 2-AP-T mismatch in the centre of the recognition site, but it does not cleave the duplexes containing 2-aminopurine in place of inner and outer G, or both. EcoRII restriction endonuclease does not cleave duplexes containing 2-aminopurine at all. The two-substrate mechanism of EcoRII-DNA interaction, however, allows hydrolysis of the duplex containing 2-aminopurine in place of adenine in the presence of the canonical substrate.     

Influence of nucleic acid base aromaticity on substrate reactivity with enzymes acting on single-stranded DNA.

Stacking between aromatic amino acids and nucleic acid bases may play an important role in the interaction of enzymes with nucleic acid substrates. In such circumstances, disruption of base aromaticity would be expected to decrease enzyme activity on the modified substrates. We have examined the requirement for DNA base aromaticity of five enzymes that act on single-stranded DNA, T4 polynucleotide kinase, nucleases P1 and S1, and snake venom and calf spleen phosphodiesterases, by comparing their kinetics of reaction with a series of dinucleoside monophosphates containing thymidine or a ring-saturated derivative. The modified substrates contained either cis-5R,6S-di-hydro-5,6-dihydroxythymidine (thymidine glycol) or a mixture of the 5R and 5S isomers of 5,6-dihydrothymidine. It was observed that for all the enzymes, except snake venom phosphodiesterase, the parent molecules were better substrates than the dihydrothymidine derivatives, while the thymidine glycol compounds were significantly poorer substrates. Snake venom phosphodiesterase acted on the unmodified and dihydrothymidine molecules at almost the same rate. These results imply that for all the remaining enzymes base aromaticity is a factor in enzyme-substrate interaction, but that additional factors must contribute to the poorer substrate capacity of the thymidine glycol compounds. The influence of the stereochemistry of the dihydrothymidine derivatives was also investigated. We observed that nuclease P1 and S1 hydrolysed the molecules containing 5R-dihydrothymidine approximately 50-times faster than those containing the S-isomer. The other enzymes displayed no measurable stereospecificity.     

Restriction endonuclease SsoII with photoregulated activity--a "molecular gate" approach

A novel method for regulating the activity of homodimeric proteins--"molecular gate" approach--was proposed and its usefulness illustrated for the type II restriction endonuclease SsoII (R.SsoII) as a model. The "molecular gate" approach is based on the modification of R.SsoII with azobenzene derivatives, which allows regulating DNA binding and cleavage via illumination with light. R.SsoII variants with single cysteine residues introduced at selected positions were obtained and modified with maleimidoazobenzene derivatives. A twofold change in the enzymatic activity after illumination with light of wavelengths of 365 and 470 nm, respectively, was demonstrated when one or two molecules of azobenzene derivatives were attached to the R.SsoII at the entrance of or within the DNA-binding site.     

The solution conformation of a carbocyclic analog of the Dickerson-Drew dodecamer: comparison with its own X-ray structure and that of the NMR structure of the native counterpart

The NMR conformation of a carbocyclic analog of the Dickerson-Drew dodecamer [d(CGC-GAAT*T*CGCG)]2 containing 6'-alpha-Me carbocyclic thymidines (T*) has been determined and compared with that of its X-ray structure. The solution structure of the 6'-alpha-Me carbocyclic thymidine modified duplex has also been compared with the solution structure of the corresponding unmodified Dickerson-Drew duplex solved by us under the same experimental conditions. The NMR structures have been based on 24 experimental distance and torsion constraints per residue for [d(CGCGAAT*T*CGCG)]2 (1) and on 21 constraints per residue for the natural counterpart. In general, both final NMR structures are more close to the B-type DNA. The cyclopentane moieties of the carbocyclic thymidine residues adopt C1'-exo B-DNA type puckers (the phase angles P = 136-139 degrees and the puckering amplitudes psi = 36-37 degrees) that are close to their previously published crystal C1'-exo or C2'-endo puckers. The main differences between the two NMR structures are for beta(T*8) and epsilon, xi(T*7) backbone torsions (27-50 degrees ), for basepair twist for the 7-8 and 8-9 basepair steps (5-6 degrees), tilt for the 8-9 step (7 degrees), roll for the 7-8 step (7 degrees), shift for the 7-8 step (0.9A) and slide for the 9-10 step (0.6A). The relatively small deviations of helical structure parameters lead to structural isomorphism of these duplexes in aqueous solutions (atomic RMSD = 1.0A). The difference of the minor groove widths (less than 0.7A) in the core part of the modified duplex in comparison with the native one is much smaller than the difference between the X-ray structures of these duplexes. A detailed comparison of NMR and X-ray structure parameters showed significant monotonic differences (0.9-2.5A) for all basepair slides in both duplexes. Deviations between NMR and X-ray structure parameters for the modified duplex were also found for basepair tilt of the 4-5 step (13 degrees), rolls for the 8-9 and 10-11 steps (16 and 19 degrees), twist of the 3-4 step (8 degrees) and shift of the 9-10 step (0.9A).     

DNA duplexes containing altered sugar residues as probes of EcoRII and MvaI endonuclease interactions with sugar-phosphate backbone

Oligonucleotides containing 1-(beta-D-2'-deoxy-threo-pentofuranosyl)cytosine (dCx) and/or 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine (dTx) in place of dC and dT residues in the EcoRII and MvaI recognition site CC(A/T)GG were synthesized in order to investigate specific recognition of the DNA sugar-phosphate backbone by EcoRII and MvaI restriction endonucleases. In 2'-deoxyxylosyl moieties of dCx and dTx, 3'-hydroxyl groups were inverted, which perturbs the related individual phosphates. Introduction of a single 2'-deoxyxylosyl moiety into a dC x dG pair resulted in a minor destabilization of double-stranded DNA structure. In the case of a dA x dT pair the effect of a 2'-deoxyxylose incorporation was much more pronounced. Multiple dCx modifications and their combination with dTx did not enhance the destabilization effect. Hydrolysis of dCx-containing DNA duplexes by EcoRII endonuclease was blocked and binding affinity was strongly depended on the location of an altered sugar. A DNA duplex containing a dTx residue was cleaved by the enzyme, but kcat/K(M) was slightly reduced. In contrast, MvaI endonuclease efficiently cleaved both types of sugar-altered substrate analogs. However it did not cleave conformationally perturbed scissile bonds, when the corresponding unmodified bonds were perfectly hydrolyzed in the same DNA duplexes. Based on these data the possible contributions of individual phosphates in the recognition site to substrate recognition and catalysis by EcoRII were proposed. We observed strikingly non-equivalent inputs for different phosphates with respect to their effect on EcoRII-DNA complex formation.     

Investigation of the inhibitory role of phosphorothioate internucleotidic linkages on the catalytic activity of the restriction endonuclease EcoRV

The inhibitory effect of phosphorothioate residues, located within one strand of double-stranded DNA, on the hydrolytic activity of the restriction endonuclease EcoRV was investigated. Specific incorporation of a phosphorothioate group at the site of cleavage yielded the sequence 5'-GATsATC-3'. This modified sequence was cleaved at a relative rate of 0.1 compared to the unmodified substrate. Substrates 5'-GATsAsTC-3' and 5'-GsATsATC-3', both containing one additional phosphorothioate substitution, were linearized at a rate of 0.04 relative to unmodified DNA. However, under the same conditions, fully dAMPS-substituted DNA was found to be virtually resistant to the hydrolytic activity of EcoRV. Further experiments showed that double-stranded DNA fragments generated by PCR containing phosphorothioate groups within both strands are potent inhibitors of EcoRV catalysis. The inhibition was independent of whether the inhibitor fragment contained an EcoRV recognition site. We concluded that substitution of the phosphate group at the site of cleavage by a phosphorothioate residue decreases the rate of EcoRV-catalyzed hydrolysis most significantly. Substitution of other phosphate groups within the recognition sequence plays a limited role in enzyme inhibition. The presence of multiple dNMPS residues at regions of the DNA removed from the EcoRV recognition site may decrease the amount of enzyme available for catalysis by nonspecific binding to EcoRV.     

DNA-duplexes with phosphoamide bond: interaction with restriction endonucleases EcoRII and SsoII

We studied the interaction of EcoRII and SsoII restriction endonucleases with synthetic DNA duplexes, containing 3'N----5'P and 3'P----5'N phosphoamide internucleotide bonds in one of the cleavage points. Enzymatic hydrolysis of the modified strand of the duplexes is blocked in all cases. The presence of phosphoamide bonds was found to reduce the rate of cleavage of the natural strand by EcoRII and to have no influence in case of SsoII. Properties of the EcoRII endonuclease complex with its substrate, containing non-cleavable 3'N----5'P internucleotide bonds in each cleavage point, were examined. In the presence of Mg2+ ions the equilibrium association constant of the enzyme-substrate complex is 3-fold reduced, and the dissociation rate constant of the complex is increased by 1.5 times.     

Identification of base-specific contacts in protein-DNA complexes by photocrosslinking and mass spectrometry: a case study using the restriction endonuclease SsoII

Specific protein-nucleic acid interactions are of paramount importance for the propagation, maintenance and expression of genetic information. Restriction endonucleases serve as model systems to study the mechanisms of DNA recognition by proteins. SsoII is a Type II restriction endonuclease that recognizes the double stranded sequence downward arrow CCNGG and cleaves it in the presence of Mg(2+)-ions, as indicated. SsoII shows sequence similarity over a stretch of approximately 70 amino acid residues with several other restriction endonucleases that recognize a similar sequence as SsoII (Cfr10I, EcoRII, NgoMIV, PspGI). In NgoMIV this stretch is involved in DNA recognition and cleavage, as shown by the crystal structure analysis of an enzyme-product complex. To find out whether the presumptive DNA recognition region in SsoII is indeed in contact with DNA we have photocrosslinked SsoII with an oligodeoxyribonucleotide in which the first guanine of the recognition sequence was replaced by 5-iodouracil. Following digestion by trypsin, the peptide-oligodeoxyribonucleotide conjugate was purified by Fe(3+)-IMAC and then incubated with hydrogen fluoride, which hydrolyzes the oligodeoxyribonucleotide to yield the peptide-deoxyuridine conjugate. The site of photocrosslinking was identified by MALDI-TOF-MS and MALDI-TOF-MS/MS to be Trp189, adjacent to Arg188, which aligns with Arg194 in NgoMIV, involved in recognition of the second guanine in the NgoMIV recognition sequence G downward arrow CCGGC. This result confirms previously published conclusions drawn on the basis of a mutational analysis of SsoII. The methodology that was employed here can be used in principle to identify the DNA binding site of any protein.     

Effects of 2-chloroadenine substitution in DNA on restriction endonuclease cleavage reactions.

The purine analog, 2-chloro-2'-deoxyadenosine triphosphate (CldATP), was incorporated enzymatically in place of dATP into the minus strand of M13mp18 duplex DNA. Its effect on protein-DNA interactions was assessed by determining the amount of DNA cleavage by type II restriction endonucleases. Substitution of chloroadenine (CIAde) for adenine (Ade) in DNA appreciably decreased the amount and rate of DNA cleavage of the minus strand when the analog was situated within the appropriate endonuclease recognition site. CIAde residues flanking a restriction site had variable effects. SmaI cleaved both CIAde-containing and control substrates with equal efficiency. NarI, however, was stimulated 1.5-fold by the presence of CIAde outside its recognition site. The effects of analog incorporation on restriction enzyme cleavage of an opposing unsubstituted strand of duplex DNA was examined by enzymatically incorporating CIdATP into the complementary minus strand of a 36-base oligonucleotide. Endonucleolytic cleavage of both plus and minus strands was reduced on 36-mers containing CIAde residues located within only the minus strand. These data suggest that CIAde residues incorporated into a single DNA strand may have an appreciable effect on DNA-protein interactions that involve one or both strands of duplex DNA.     

Is Thymidine Glycol Containing DNA a Substrate of E. coli DNA Mismatch Repair System?

The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active “sliding clamp” conformation on Tg-DNA is problematic.     

Covalent binding of Cys142 from SsoII methyltransferase with DNA duplexes, containing a phosphoryldisulfide internucleotide group

DNA duplexes containing a single phosphoryldisulfide link in place of the natural internucleotide phosphodiester bond were employed in affinity modification of Cys142 in cytosine-C5 DNA methyltransferase SsoII (M.SsoII). The possibility of duplex-M.SsoII conjugation as a result of disulfide exchange was demonstrated. The crosslinking efficiency proved to depend on the DNA primary structure, modification position, and the presence of S-adenosyl-L-homocysteine, a nonreactive analog of the methylation cofactor. The SH group of M.SsoII Cys142 was assumed to be close to the DNA sugar-phosphate backbone in the DNA-enzyme complex.     

Site-specifically located 8-amino-2'-deoxyguanosine: thermodynamic stability and mutagenic properties in Escherichia coli

2-Nitropropane (2-NP), an important industrial solvent and a component of cigarette smoke, is mutagenic in bacteria and carcinogenic in rats. 8-Amino-2'-deoxyguanosine (8-amino-dG) is one of the types of DNA damage found in liver, the target organ in 2-NP-treated rats. To investigate the thermodynamic properties of 8-amino-dG opposite each of the four DNA bases, we have synthesized an 11mer, d(CCATCG*CTACC), in which G* represents the modified base. By annealing a complementary DNA strand to this modified 11mer, four sets of duplexes were generated each containing one of the four DNA bases opposite the lesion. Circular dichroism studies indicated that 8-amino-dG did not alter the global helical properties of natural right-handed B-DNA. The thermal stability of each duplex was examined by UV melting measurements and compared with its unmodified counterpart. For the unmodified 11mer, the relative stability of the complementary DNA bases opposite G was in the order C > T > G > A, as determined from their -DeltaG degrees values. The free energy change of each modified duplex was lower than its unmodified counterpart, except for the G*:G pair that exhibited a higher melting transition and a larger -DeltaG degrees than the G:G duplex. Nevertheless, the stability of the modified 11mer duplex also followed the order C > T > G > A when placed opposite 8-amino-dG. To explore if 8-amino-dG opposite another 8-amino-dG has any advantage in base pairing, a G*:G* duplex was evaluated, which showed that the stability of this duplex was similar to the G*:G duplex. Mutagenesis of 8-amino-dG in this sequence context was studied in Escherichia coli, which showed that the lesion is weakly mutagenic (mutation frequency approximately 10(-3)) but still can induce a variety of targeted and semi-targeted mutations.     

Cleavage by restriction endonucleases MvaI and EcoRII of substrates modified in amino groups of heterocyclic bases

14-membered DNA-duplexes containing modified nucleoside residues, viz 4-N-methyldeoxycytidine (m4dC), 6-N-methyldeoxyadenosine (m6dA) or deoxyinosine (dI), in only one strand of the recognition site (CCA/TGG) of MvaI and EcoRII endonucleases were synthesized. It was shown that MvaI and EcoRII endonucleases interact with the exocyclic amino groups of the external dC residues and of the central dA residue of the recognition site exposed into the DNA major groove. These endonucleases which are isochizomers were found to possess different mechanisms of substrate cleavage. The ability of MvaI endonuclease to hydrolyze only unmodified strand of methylated duplexes allows one to make site-directed single-strand nicks in double-stranded DNA. Elimination of the 2-NH2-group located in the minor groove of DNA by substituting dI for dG had little, if any, effect on the hydrolytic activity of EcoRII and MvaI endonucleases.     

Cleavage of concatamer-type substrates by restriction endonucleases MVA1 and SSO1I

The interaction of enzymes SsoII (decreases CCNGG) and MvaI (CC decreases A/TGG) with concatemeric DNA duplexes used earlier to study EcoRII (decreases CCA/TGG) TGG was investigated with a view of elucidating the general principles of the restriction endonuclease function. A pattern common for all the three enzymes was observed with DNA duplexes containing AA or TT pairs in the central position of the recognition site. The AA pair blocks or substantially hinders the endonuclease action, whereas the TT pair is either less inhibitory or altogether inert. SsoII, similar to EcoRII was able to processively cleave the concatemeric substrates and to interact with (or to be close to) the hydrogen in the 5th position of the outer dC residue of the recognition site. MvaI was found to differ from EcoRII in the way they recognize and cleave the same nucleotide sequence. The substrate-bound MvaI molecule is incapable of linear diffusion along the DNA. Effective hydrolysis of dU- and m5dC-containing polymers rules out the participation of hydrophobic contacts of the enzyme with the methyl group of the dT residue and with the 5th hydrogen of the outer dC residue of the recognition site in DNA-protein interactions.