Nuclear magnetic resonance studies of cis-syn, trans-syn, and 6-4 photodimers of thymidylyl(3'-5')thymidine monophosphate and cis-syn photodimers of thymidylyl(3'-5')thymidine cyanoethyl phosphotriester

Three out of four possible photodimers of thymidylyl(3'-5')thymidine monophosphates (i.e., cis-syn, 6-4, and one of the trans-syn) and two structural isomers (i.e., R and S forms) of cis-syn-thymidylyl(3'-5')thymidine cyanoethyl phosphotriester have been isolated and purified from the reaction mixtures after UV irradiation and studied by multinuclear magnetic resonance Spectroscopy. All five inter thymine base linked photodimers have grossly similar structures which are quite different from those of the parent thymidylyl(3'-5')thymidine. The base of Tp- is in the syn conformation, and that of -pT it is in the anti conformation. The sugar puckering of Tp- is dominated by the 2E conformer, but in -pT it is in 4E; except for the conformer around C5'-O5' bond, the 6-4 isomer is very similar to those of cis-syn and trans-syn conformation. As expected, there are sugar-phosphate backbone distortions in the phosphotriesters, due to the neutralization of the negative charge of the phosphate. In general the structures of all five photodimers are very close to those of the cis-syn photodimer of thymidylyl(3'-5')thymidine monophosphate cyanoethyl ester as studied by X-ray diffraction [Cadet, J., Voituriez, L., Hruska, F. E., & Grand, A. (1985) Biopolymers 24, 897-903; Hruska, F. E., Voituriez, L., Grand, A., & Cadet, J. (1986) Biopolymers 25, 1401-1417]. While the trans-syn photodimer has two structural isomers, only one [C6(of Tp-)-R] was produced by the UV irradiation and studied.     

Conformational variations of the cis-syn cyclobutane-type photodimer in DNA and RNA.

The recent NMR study of a cis-syn photodimer B-DNA 10mer-duplex (Taylor et al., Biochemistry 29, 8858 (1990)) showed the cyclobutane (CB) ring with a puckered-twist in a right-handed sense (CB+). This is opposite to that of the crystal structure of cis-syn d-TpT(cyano-ethyl)(d-T[p]T-CE) which has a left-handed puckered-twist (CB-)(Hruska et al., Biopolymers 25, 1399 (1986)). 2D-NOESY experiments were performed on cis-syn d-T[p]T and cis-syn U[p]U at 25 and 35 degrees C, respectively, to investigate the puckering mode of the cyclobutane ring of isolated cis-syn photodimers of the DNA and RNA types. The DNA photodimers showed interconversion of the puckered-twist of the cyclobutane ring between CB- and CB+ and interconversion of the glycosidic angle between syn and anti in both nucleoside residues. Interestingly, in the RNA photodimer only the CB- puckering mode with syn conformation of the glycosidic angle of the U[p]- was observed. These different dynamical behaviors of the photodimer in DNA and RNA might portend differential conformational effects on their corresponding normal nucleic acid regions. In addition these results indicate differences in the cyclobutane ring conformation of the cis-syn d-T[p]T, not only in solution and crystalline states, but also when the dimer is isolated and in duplex forms.     

1H NMR assignment and melting temperature study of cis-syn and trans-syn thymine dimer containing duplexes of d(CGTATTATGC).d(GCATAATACG)

The preparation and spectroscopic characterization of duplex decamers containing site-specific cis-syn and trans-syn thymine dimers are described. Three duplex decamers, d(CGTATTATGC).d(GCATAATACG), d(CGTAT[c,s]TATGC).d(GCATAATACG), and d(CGTAT[t,s]TATGC).d(GCATAATACG), were prepared by solid-phase phosphoramidite synthesis utilizing cis-syn and trans-syn cyclobutane thymine dimer building blocks (Taylor et al., 1987; Taylor & Brockie, 1988). NMR spectra (500 MHz 2D 1H and 202 MHz 1D 31P) were obtained in "100%" D2O at 10 degrees C, and 1D exchangeable 1H spectra were obtained in 10% D2O at 10 degrees C. 1H NMR assignments for H5, H6, H8, CH3, H1', H2', and H2" were made on the basis of standard sequential NOE assignment strategies and verified in part by DQF COSY data. Comparison of the chemical shift data suggests that the helix structure is perturbed more to the 3'-side of the cis-syn dimer and more to the 5'-side of the trans-syn dimer. Thermodynamic parameters for the helix in equilibrium coil equilibrium were obtained by two-state, all or none, analysis of the melting behavior of the duplexes. Analysis of the temperature dependence of the T5CH3 1H NMR signal gave delta H = 44 +/- 4 kcal and delta S = 132 +/- 13 eu for the trans-syn duplex. Analysis of the concentration and temperature dependence of UV spectra gave delta H = 64 +/- 6 kcal and delta S = 178 +/- 18 eu for the parent duplex and delta H = 66 +/- 7 kcal and delta S = 189 +/- 19 eu for cis-syn duplex. It was concluded that photodimerization of the dTpdT unit to give the cis-syn product causes little perturbation of the DNA whereas dimerization to give the trans-syn product causes much greater perturbation, possibly in the form of a kink or dislocation at the 5'-side of the dimer.     

Intramolecular H atom abstraction from the sugar moiety by thymine radicals in oligo- and polydeoxynucleotides

Hydroxyl radical addition to uracil (U) has been suggested to lead to strand breaks in polyuridylic acid, an occurrence attributed in part to H atom abstraction by .U-OH radicals from the ribose moiety [D.G.E. Lemaire et al., Int. J. Radiat. Biol. 45, 351-358 (1984)]. We have investigated this particular reaction by means of the hydroxyl radical-induced products of thymine (T), pT, TpT, TpTpT, polythymidylic acid (poly-T), (T + dR) poly-dA.poly-T, and a mixture of T and 2-deoxyribose (dR). The major monomeric product of .T-OH in TpT, TpTpT, poly-T, and poly-dA.poly-T was found to be 5-hydroxy-6-hydrothymine (H-T-OH), while that in T, pT, and T plus dR was thymine glycol (HO-T-OH). These results indicated that the intramolecular H atom abstraction from a nearby sugar (in this case, deoxyribose) moiety by base radicals, i.e., .T-OH, occurs in oligo- and polydeoxynucleotides of T. In poly-T, the yield of H-T-OH is not much greater than in TpT or TpTpT, indicating that the abstraction of an H atom from the sugar moiety of a nucleotide subunit further than two nucleotides along the chain may not be significant. Additionally, a corresponding decrease in the yield of HO-T-OH with an increase in the yield of H-T-OH suggests that the formations of these two types of thymine products are competitive.     

Reaction of Escherichia coli and yeast photolyases with homogeneous short-chain oligonucleotide substrates

Similar rates have been observed for dimer repair with Escherichia coli photolyase and the heterogeneous mixtures generated by UV irradiation of oligothymidylates [UV-oligo(dT)n, n greater than or equal to 4] or DNA. Comparable stability was observed for ES complexes formed with UV-oligo(dT)n, (n greater than or equal to 9) or dimer-containing DNA. In this paper, binding studies with E. coli photolyase and a series of homogeneous oligonucleotide substrates (TpT, TpTp, pTpT, TpTpT, TpTpT, TpTpTpT, TpTpTpT, TpTpTpT, TpTpTpT) show that about 80% of the binding energy observed with DNA as substrate (delta G approximately 10 kcal/mol) can be attributed to the interaction of the enzyme with a dimer-containing region that spans only four nucleotides in length. This major binding determinant (TpTpTpT) coincides with the major conformational impact region of the dimer and reflects contributions from the dimer itself (TpT, delta G = 4.6 kcal/mol), adjacent phosphates (5'p, 0.8 kcal/mol; 3'p, 1.1 kcal/mol), and adjacent thymine residues (5'T, 0.8 kcal/mol; 3'T, 1.3 kcal/mol). Similar turnover rates (average kcat = 6.7 min-1) are observed with short-chain oligonucleotide substrates and UV-oligo(dT)18, despite a 25,000-fold variation in binding constants (Kd). In contrast, the ratio Km/Kd decreases as binding affinity decreases and appears to plateau at a value near 1. Turnover with oligonucleotide substrates occurs at a rate similar to that estimated for the photochemical step (5.1 min-1), suggesting that this step is rate determining. Under these conditions, Km will approach Kd when the rate of ES complex dissociation exceeds kcat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational features of DNA containing a cis-syn photodimer

In an effort to understand the conformational and structural changes in DNA brought about by thymine photodimer, computer modeling and molecular mechanics energy calculations were performed on DNA hexamer and dodecamer duplexes containing a cis-syn photodimer. The conformation of the crystal structure of the cyanoethyl phosphate ester of the thymine dimer (Hruska et al., Biopolymers 25, 1399-1417 (1986)) was used in modeling the photodimer portion. Various starting conformations were used in the modeling procedure and the structures were minimized both retaining and later relaxing the crystallographic geometry of the cyclobutane ring. The results indicate that most of the deformation is restricted to the thymine dimer region, and that the conformational changes decrease rapidly on either side of the region containing the photodimer. The structural changes brought about by the introduction of the photodimer can be accommodated within six base paired duplex without significant bend in the DNA. More conformational changes are observed on the 5'-side of the photodimer than on the 3'-side. The conformational features, such as backbone torsion angles and sugar puckers, of the energy minimized structures are discussed in the context of the solution structures determined by NMR on a series of oligomers containing photodimers (Rycyna et al., Biochemistry 27, 3152-3163 (1988)).     

Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. II. Phosphorylation by phage T4 polynucleotide kinase

Phage T4 polynucleotide kinase (EC 2.7.1.78) proved incapable of catalyzing the phosphorylation of thymidylyl-(3'----5')-thymidine containing either a cis-syn-cyclobutane pyrimidine dimer (d-T less than p greater than T) or a 6-4'-[pyrimidin-2'-one]pyrimidine photoproduct (d-T[p]-T), and similarly the UV-modified compounds of (dT)3 bearing either photoproduct at their 5'-end (d-T less than p greater than TpT and d-T[p]TpT). In contrast, the 3'-structural isomers of these trinucleotides (d-TpT less than p greater than T and d-TpT[p]T) were phosphorylated at the same rate as the parent compound. These phosphorylatable lesion-containing oligonucleotides are quantitatively released from UV-irradiated poly(dA):poly(dT) by enzymatic hydrolysis with snake venom phosphodiesterase and alkaline phosphatase (Liuzzi, M., Weinfeld, M., and Paterson, M. C. (1989) J. Biol. Chem. 264, 6355-6363). By combining this digestion regimen with phosphorylation by polynucleotide kinase and [gamma-32P]ATP, pyrimidine dimers were quantitated at the fmol level following exposure of poly(dA):poly(dT) and herring sperm DNA to biologically relevant UV fluences. The rate of dimer induction in the synthetic polymer, approximately 10 dimers/10(6) nucleotides/Jm-2, was in close agreement with that obtained by conventional methods. Dimers were induced at one-fourth of this rate in the natural DNA. Further treatment of the phosphorylated oligonucleotides derived from irradiated herring sperm DNA with nuclease P1 released the labeled 5'-nucleotide, thus permitting analysis of the nearest-neighbor bases 5' to the lesions. We observed a ratio for pyrimidine-to-purine bases of almost 6:1, implicating tripyrimidine stretches as hotspots for UV-induced DNA damage.     

Cyclobutane thymine dimers in a ras proto-oncogene hot spot activate the gene by point mutation.

ras proto-oncogenes with a cyclobutane-type thymine photodimer (cis-syn or trans-syn isomer) were constructed by replacement of a portion of the gene with a chemically synthesized fragment. When the genes were transfected by the calcium phosphate method into mouse NIH3T3 cells, they induced focus-formation, indicating that both photoproducts were mutagenic in mammalian cells. Sequence analysis of the ras gene fragments derived from the transformed cells showed that the genes were activated by a point mutation. The mutations detected most frequently were 3'-T-->A for the cis-syn isomer and 5'-T-->A for the trans-syn isomer. In contrast, a different trend of mutations was observed when a primer on a DNA template with a cis-syn dimer was extended in vitro by either DNA polymerase beta or alpha.     

Solid phase-supported thymine dimers for the construction of dimer-containing DNA by combined chemical and enzymatic synthesis: a potentially general method for the efficient incorporation of modified nucleotides into DNA.

The ability to study the structure-activity relationships of the cis-syn thymine dimer, the major photoproduct of DNA, has been greatly aided by the availability of a building block suitable for its sequence-specific incorporation into oligonucleotides by standard automated DNA synthesis. Unfortunately, its usefulness is compromised by the fact that it takes six steps to synthesize in low overall yield and, as with all phosphoramidite building blocks, has to be used in great excess over the support in standard automated synthesis. To extend the usefulness of this building block, we have directly coupled it to standard A, C, G and T long chain alkylamine-linked controlled pore glass supports to yield a solid phase-supported dimer. We then demonstrate that 13mers containing a 3'-terminal d(T[cis-syn]TN) group synthesized with this support at 0.2 micromol scale can be efficiently incorporated into longer oligonucleotides by both primer extension with 3'-->5'exonuclease-deficient Klenow fragment or T4 polymerase and dNTPs or by enzymatic ligation with T4 DNA ligase to another oligonucleotide opposite a complementary template. The site specificity and integrity of the cis-syn thymine dimer after both primer extension and ligation was confirmed by cis-syn dimer-specific cleavage with T4 denV endonuclease V. This general approach should be applicable to the synthesis of many types of site-specific nucleic acid modifications and would be of particular use for those for which the required building blocks are expensive or difficult to make.     

Antigen structural requirements for recognition by a cyclobutane thymine dimer-specific monoclonal antibody.

A monoclonal antibody (TDM-2) specific to a UV-induced cyclobutane pyrimidine dimer (T[cis-syn]T) has previously been established; however,the immunization had used UV-irradiated calf-thymus DNA containing a heterogeneous mixture of photoproduct sites. We investigated here the structural requirements of antigen recognition by the antibody using chemically synthesized antigen analogs. TDM-2 bound with cis-syn,but not trans-syn thymine dimer,and could bind strongly with four nucleotide analogs in which the cis-syn pyrimidine dimer was located in the center. Antigen analogs containing abasic linkers at the 5'- or 3'-side of the cis-syn cyclobutane pyrimidine dimer were synthesized and tested for binding to TDM-2. The results indicated that TDM-2 recognizes not only the cyclobutane ring but also both the 5'- and 3'-side nucleosides of the cyclobutane dimer. Furthermore,it was proved that either the 5'- or 3'-side phosphate group at a cyclobutane dimer site was absolutely required for the affinity to TDM-2. The antibody showed a strong binding to single stranded DNA but indicated little binding to double stranded DNA.     

Conformational changes in the oligonucleotide duplex d(GCGTTGCG) x d(CGCAACGC) induced by formation of a cis-syn thymine dimer. A two-dimensional NMR study

In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCGTTGCG) x d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two-dimensional nuclear Overhauser enhancement (2D NOE) spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy have been applied to assign all the non-exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis-syn cyclobutane-type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the 1H chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B-DNA structure, the main conformational change occurring at the site of the thymine dimer.     

In vitro evidence that UV-induced frameshift and substitution mutations at T tracts are the result of misalignment-mediated replication past a specific thymine dimer.

A previous study of UV-induced (254 nm) mutations in the lacI gene of Escherichia coli found that frameshift mutations accounted for about 35% of the observed mutations and that these mutations occurred predominantly at An.Tn sequences [Miller, J.H. (1985) J. Mol. Biol. 182, 48-65]. Because An.Tn sequences are hotspots for cis-syn thymine dimer formation [Brash, D.E., & Haseltine, W. A. (1982) Nature 298, 189-192], it would appear that UV-induced frameshift mutations are the result of an error during replicative bypass of a thymine dimer within such a sequence. To test the validity of such a proposal, replication experiments were carried out on templates containing cis-syn thymine dimers at each of the five possible sites of a T6 tract. The 59-mer templates were prepared by ligating oligonucleotides containing an EcoRI site to the 5'-end of decamers containing the cis-syn thymine dimer and oligonucleotides containing the primer site to the 3'-end. Primer-extension reactions were then carried out on these templates with a 3'----5' exonuclease-deficient (exo-) Klenow fragment of E. coli polymerase I and an exo-T7 polymerase (Sequenase Version 2.0). The replicative bypass products were cleaved with EcoRI to rigorously establish and quantify the presence of frameshift mutations. Both polymerases were able to bypass dimers at all sites, but only the exo-T7 polymerase led to detectable frameshifts, both -1 (approximately 30%) and -2 (approximately 5%), and only with the template containing a cyclobutane dimer at the second site from the 5'-end of the T6 tract. Sequencing of the T7 polymerase-catalyzed bypass products of all templates demonstrated that within the limits of discrimination only As were introduced opposite the dimer-containing T tracts. The only exception was for the template with the dimer at the second site which led to a readily detectable amount of a substitution mutation (approximately 30%) opposite the 5'-thymine of the T6 tract. A mechanism involving a competition between reversible misalignment and realignment steps and irreversible elongation steps is proposed to explain the origin of both the frameshift and the substitution mutations. The implications of this work to the mechanism of UV-induced frameshift and substitution mutations at T tracts in vivo are discussed.     

Proton magnetic resonance study of nucleosides,nucleotides, and dideoxynucleoside monophosphates containing a syn pyrimidine base

Proton magnetic resonance data have been obtained for 6-methyl-2′-deoxyuridine (dT*), its 3′- and 5′-monophosphates, and its 3′,5′-diphosphate, as well as for the corresponding thymine derivatives. The synthesis of the dideoxynucleoside monophosphates—d(TpT), d(T*pT), d(TpT*), and d(T*pT*)—was accomplished, and spectral data were obtained for these four dimers. The data show that the 6-methyluracil base prefers the syn conformation about the N-glycosyl bond at the monomer and dimer levels. The presence of the syn base leads to increases in the cis couplings of the sugar ring, J_1′2″ and J_2′3′, which indicate a trend towards eclipsing of the substituents on the C1′-C2′ and C2′-C3′ fragments. This trend is discussed in terms of changes in the pseudorotational parameters which describe the pucker of the ring. The syn base destabilizes the g⁺ conformer about the C4′-C5′ bond, leading to a preference for the t conformer in all dT* residues at the monomer and dimer levels. Preliminary work on the formation of cyclobutane-type photodimers in d(T*pT) and d(T*pT*) is discussed and presented as evidence for the capability of the syn 6-methyluracil base to form base-stacked complexes.     

Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. I. Nuclease P1-mediated hydrolysis of the intradimer phosphodiester linkage

Our recent findings suggest that enzymatic hydrolysis of the intradimer phosphodiester bond may constitute the initial step in the repair of UV light-induced cyclobutane pyrimidine dimers in human cells. To examine the susceptibility of this phosphodiester linkage to enzyme-mediated hydrolysis, the trinucleotide d-Tp-TpT was UV-irradiated and the two isomeric compounds containing a cis-syn-cyclobutane dimer were isolated by high performance liquid chromatography and treated with various deoxyribonucleases. Snake venom phosphodiesterase hydrolyzed only the 3'-phosphodiester group in the 5'-isomer (d-T less than p greater than TpT) but was totally inactive toward the 3'-isomer (d-TpT less than p greater than T). In contrast, calf spleen phosphodiesterase only operated on the 3'-isomer by cleaving the 5'-internucleotide bond. Kinetic analysis revealed that (i) the activity of snake venom phosphodiesterase was unaffected by a dimer 5' to a phosphodiester linkage, (ii) the action of calf spleen phosphodiesterase was partially inhibited by a dimer 3' to a phosphodiester bond, and (iii) Escherichia coli phr B-encoded DNA photolyase reacted twice as fast with d-T less than p greater than TpT as with d-TpT less than p greater than T. Mung bean nuclease, nuclease S1, and nuclease P1 all cleaved the 5'-internucleotide linkage, but not the intradimer phosphodiester bond, in d-TpT less than p greater than T. Both phosphate groups in d-T less than p greater than TpT were refractory to mung bean nuclease or nuclease S1. Incubation of d-T less than p greater than TpT with nuclease P1, however, generated the novel compound dT less than greater than d-pTpT containing a severed intradimer phosphodiester linkage. Accordingly, nuclease P1 represents the first purified enzyme known to hydrolyze an intradimer phosphodiester linkage.     

Synthesis of a trans-syn thymine dimer building block. Solid phase synthesis of CGTAT[t,s]TATGC.

The synthesis of a building block for the sequence specific introduction of the trans-syn thymine dimer into oligonucleotides via solid phase DNA synthesis technology is described. CGTAT[t,s]TATGC was synthesized in 48% overall yield by a partially automated procedure. The stepwise coupling yield for addition of the trans-syn thymine dimer building block was 58%. The dimer containing oligonucleotide was characterized by 500 MHz 1H COSY and NOESY spectroscopy and 202.5 MHz 31P NMR. The 1H chemical shifts for the trans-syn thymine dimer unit of the decamer were found to be quite similar to those found for the trans-syn thymine dimer of TpT. Upon photolysis at 254 nm, CGTAT[t,s]TATGC was converted to a major product which coeluted with authentic CGTATTATGC and a minor product which coeluted with authentic CGTAT[c,s]TATGC, further supporting the presence of an intact trans-syn thymine dimer unit.     

A comparative conformational study of thymidylyl(3'----5')-thymidine, thymidylyl(3'----5')-5'-thio-5'- deoxythymidine and thymidinylacetamido-[3'(O)----5'(C)]-5'-deoxythymidine

A comparative 270 MHz NMR spectroscopic study on the solution structure of the dimer d(TpT) 1, and its two analogues, namely, d(TpST) 2, and NH2d(TcmT) 4 has been reported. Analysis of chemical shifts and coupling constants indicate that: (i) The sugar moieties of the constituent nucleotides are not affected by modification of the internucleotide linkages and adopt preferentially an S-type conformation. (ii) The C4'-C5' bond in the pT part of the modified dimers 2 and 4 shows a large conformational freedom (gamma+ = 32% and 35%, respectively) compared to 1 (gamma+ = 75%). (iii) The population of the trans conformer about C5'-O5' is less important in d(TpST) 2 compared to d(TpT) 1. (iv) The C3'-O3' bond in 2 adopts a trans conformation as in 1. (v) The glycosidic bonds in the modified dimers 2 and 4 showed preferential syn conformation. UV and CD data show that the modified dimers 2 and 4 have poor tendency to stack intramolecularly, they also base pair less efficiently with d(ApA) as compared to d(TpT) 1.     

Substituent effects on the puckering mode of the cyclobutane ring and the glycosyl bond of cis–syn photodimers

The cyclobutane ring (CB) puckering of a cis–syn DNA photodimer (cis–syn d-T[p]T) differs from that of a cis–syn RNA photodimer (cis–syn r-U [p] U) [J.-K. Kim and J. L. Alderfer (1992) Journal of Biomolecular Structure and Dynamics, Vol. 9 , p. 1705]. In cis–syn d-T [p] T, interconversion of the CB ring between CB⁺ and CB^? is observed, while in cis–syn r-U [p] U only CB^? is observed. In the CB⁺ conformation, the two thymine rings of the dimer are twisted in a right-handed fashion, as are the bases in B-form DNA. In case of CB^? they are twisted in a left-handed fashion. The C5 (base) and/or C2′ (sugar) substituents apparently affect the CB ring flexibility in cis–syn d-T [p] T and cis–syn r-U [p] U. To study the effects of the C5 substituent on CB ring flexibility, two-dimensional nuclear Overhauser effect (NOE) and ³¹P-nmr experiments were performed on cis–syn d-T [p] U, cis–syn d-U [p] T, and cis–syn d-U [p] U photodimers to investigate the CB puckering mode and overall molecular conformation and dynamics. The NOE results indicate the 5-methyl group in the photodimer induces conformational flexibility of the CB ring. In cis–syn d-T [p] U and cis–syn d-U [p] T, both CB⁺ and CB^? puckering modes are observed. This indicates interconversion between two modes takes place as observed in cis–syn d-T [p] T. In the case of cis–syn d-U [p] U, only the puckering CB^? mode is observed. All three DNA-type dimers—cis–syn d-T [p] U, cis–syn d-U [p] T, cis–syn d-U [p] U—show a characteristic flexibility of glycosidic bonds at the 5′ residue; cis–syn d-T [p] T demonstrates syn–anti interconversion for both the 3′ and 5′ sides, while the others are exclusively anti on the 3′ side. In contrast, the ribophotodimer, cis–syn r-U [p] U, lacking the C5 methyls and having a C2′-OH, demonstrates no conformational flexibility in the CB ring or in either of the glycosidic bonds. Differential flexibility of the three DNA-type dimers (cis–syn d-T [p] U, cis–syn d-U [p] T, cis–syn d-U [p] U) and the RNA dimer (cis–syn r-U [p] U) in the sugar-phosphate backbone region is also apparent from the temperature dependence of the ³¹P chemical shifts of these photodimers compared to their normal dimer analogues. Over the temperature range 18-63°C, the chemical shift change is reduced 22–42% in three DNA-type dimers, while it is reduced 71% in cis–syn r-U [p] U, suggesting the RNA-type dimer is more rigid. © 1993 John Wiley & Sons, Inc.     

cis-syn thymine dimers are not absolute blocks to replication by DNA polymerase I of Escherichia coli in vitro

Both Escherichia coli DNA polymerase I (pol I) and the large fragment of pol I (Klenow) were found to bypass a site-specific cis-syn thymine dimer, in vitro, under standard conditions. A template was constructed by ligating d(pCGTAT[c,s]TATGC), synthesized via a cis-syn thymine dimer phosphoramidite building block, to a 12-mer and 19-mer. The site and integrity of the dimer were verified by use of T4 denV endonuclease V. Extension of a 15-mer on the dimer-containing template by either pol I or Klenow led to dNTP and polymerase concentration dependent formation of termination and bypass products. At approximately 0.15 unit/microL and 1-10 microM in each dNTP, termination one prior to the 3'-T of the dimer predominated. At 100 microM in each dNTP termination opposite the 3'-T of the dimer predominated and bypass occurred. Bypass at 100 microM in each dNTP depended on polymerase concentration, reaching a maximum of 20% in 1 h at approximately 0.2 unit/microL, underscoring the importance of polymerase binding affinity for damaged primer-templates on bypass. Seven percent bypass in 1 h occurred under conditions of 100:10 microM dATP:dNTP bias, 1% under dTTP bias, and an undetectable amount under either dGTP or dCTP bias. At 100 microM in each dNTP, the ratio of pdA:pdG:pdC:pdT terminating opposite the 3'-T of the dimer was estimated to be 37:25:10:28. Sequencing of the bypass product produced under these conditions demonstrated that greater than 95% pdA was incorporated opposite both Ts of the dimer and that little or no frame shifting took place.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H NMR study of the exchangeable protons of the duplex d(GCGTTGCG).d(CGCAACGC) containing a thymine photodimer.

A comparison is presented of the imino proton NMR spectra of the double stranded octamer d(GCGTTGCG).d(CGCAACGC) and the same octamer in which the two central thymine residues occur as a cis-syn thymine dimer. Except for the terminal base pairs all imino protons were detected and assigned in the NMR spectrum. The spectra show that in the thymine dimer duplex, contrary to common belief, all base pairs occur in a hydrogen bonded form, although the hydrogen bonds of the two central AT base pairs are substantially weakened. The melting temperature decreases about 13 degrees C on thymine dimer formation.     

Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7

Ultraviolet-induced DNA damage poses a lethal block to replication. To understand the structural basis for this, we determined crystal structures of a replicative DNA polymerase from bacteriophage T7 in complex with nucleotide substrates and a DNA template containing a cis-syn cyclobutane pyrimidine dimer (CPD). When the 3' thymine is the templating base, the CPD is rotated out of the polymerase active site and the fingers subdomain adopts an open orientation. When the 5' thymine is the templating base, the CPD lies within the polymerase active site where it base-pairs with the incoming nucleotide and the 3' base of the primer, while the fingers are in a closed conformation. These structures reveal the basis for the strong block of DNA replication that is caused by this photolesion.