DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer

Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis-syn-cyclobutane thymine dimer (T[c,s]T). ³¹P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[c,s]T) and d(TAT[c,s]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[c,s]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V_H domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3′-side of the nucleotides onto the H1 and H3 segments, with the 5′-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.     

Role of human DNA polymerase κ in extension opposite from a cis-syn thymine dimer

Exposure of DNA to UV radiation causes covalent linkages between adjacent pyrimidines. The most common lesion found in DNA from these UV-induced linkages is the cis-syn cyclobutane pyrimidine dimer. Human DNA polymerase κ (Polκ), a member of the Y-family of DNA polymerases, is unable to insert nucleotides opposite the 3'T of a cis-syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 3'T of the dimer by another DNA polymerase. We present here the structure of human Polκ in the act of inserting a nucleotide opposite the 5'T of the cis-syn T-T dimer. The structure reveals a constrained active-site cleft that is unable to accommodate the 3'T of a cis-syn T-T dimer but is remarkably well adapted to accommodate the 5'T via Watson-Crick base pairing, in accord with a proposed role for Polκ in the extension reaction opposite from cyclobutane pyrimidine dimers in vivo.     

U-U and T-T cyclobutane dimers have different mutational properties.

We have examined the mutagenic properties in E. coli of single stranded vectors containing a uniquely placed cis-syn or trans-syn uracil-uracil cyclobutane dimer in the sequence 5' GCAAGUUGGAG 3', and compared these with the properties of the corresponding T-T dimers in the same sequence context. The frequencies with which U-U and T-T photoproducts were bypassed were similar in SOS induced cells, and each induced similar frequencies of mutations. However, although both U-U and T-T cis-syn dimers showed a preference for misincorporation in about 5-7% of the replication products, with T or G being incorporated in place of A, the ratios of these events differed, being > 4:1 for T-T cis-syn, but only 2:1 for U-U cis-syn. A shift towards G insertion opposite dimerized uracil was also found with the trans-syn dimers, but the difference was greater; T and G were misincorporated opposite the U-U trans-syn dimer in a ratio of 1:2, compared with 4:1 for its T-T counterpart. In addition, the U-U dimer induced only nucleotide substitutions, unlike the T-T photoproduct which induced single nucleotide deletions as well as substitutions. We conclude that even relatively minor differences in photoproduct structure, such as the presence of a methyl group at C-5, can alter mutational properties, and that such properties cannot depend only on the attributes of the DNA polymerase. Neither the efficiency of bypass, the error frequency nor the mutation spectrum of either U-U isomer is influenced by DNA uracil glycosylase. In vitro, the U-U cis-syn dimer is a substrate for DNA photolyase, but not for the glycosylase.     

Preparation and characterization of DNA containing a site-specific nonadjacent cyclobutane thymine dimer of the type implicated in UV-induced -1 frameshift mutagenesis.

One mechanism for the origin of UV-induced -1 deletion mutations involves the bypass of a nonadjacent cis-syn cyclobutane pyrimidine dimer containing a single intervening nucleotide. To begin to investigate this mechanism, we required a method for obtaining a single, site-specific, nonadjacent dimer. One approach to the preparation of a nonadjacent dimer is to irradiate a DNA duplex containing a centrally located TNT sequence in which the two T's are paired to an AA sequence in an otherwise fully complementary strand. Triplet-sensitized irradiation of the duplex formed between the 13-mer d(GAGTATCTATGAG) and the 12-mer d(CTCATAATACTC) on ice gave a major product that could be reverted to the parent 13-mer by 254 nm irradiation. Proton NMR experiments established the major product to be the nonadjacent cis-syn cyclobutane dimer formed between the two T's of the TCT sequence. Melting temperature studies show that the nonadjacent dimer is more destabilizing to DNA duplex structure than a normal cis-syn dimer and is as stable as the parental bulged DNA duplex. The nonadjacent dimer-containing 13-mer was ligated into a 51-mer and used as a template for primer-extension studies by DNA polymerases. The nonadjacent dimer could not be bypassed by Sequenase Version 2.0 and terminated synthesis primarily prior to and opposite the 3'-T of the dimer. In contrast, approximately 30% of the dimer was bypassed by an exonuclease-deficient (exo-) Klenow fragment, and termination occurred primarily opposite the 3'- and 5'-T's of the dimer. Bypass of the nonadjacent dimer by exo(-) Klenow fragment led primarily to a single-nucleotide deletion mutation as well as small amounts of a full-length product and a four-nucleotide deletion that could be explained by a primer misalignment mechanism.     

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.     

Photosensitized [2 + 2] cycloaddition of N-acetylated cytosine affords stereoselective formation of cyclobutane pyrimidine dimer

Photocycloaddition between two adjacent bases in DNA produces a cyclobutane pyrimidine dimer (CPD), which is one of the major UV-induced DNA lesions, with either the cis-syn or trans-syn structure. In this study, we investigated the photosensitized intramolecular cycloaddition of partially-protected thymidylyl-(3'→5')-N(4)-acetyl-2'-deoxy-5-methylcytidine, to clarify the effect of the base modification on the cycloaddition reaction. The reaction resulted in the stereoselective formation of the trans-syn CPD, followed by hydrolysis of the acetylamino group. The same result was obtained for the photocycloaddition of thymidylyl-(3'→5')-N(4)-acetyl-2'-deoxycytidine, whereas both the cis-syn and trans-syn CPDs were formed from thymidylyl-(3'→5')-thymidine. Kinetic analyses revealed that the activation energy of the acid-catalyzed hydrolysis is comparable to that reported for the thymine-cytosine CPD. These findings provided a new strategy for the synthesis of oligonucleotides containing the trans-syn CPD. Using the synthesized oligonucleotide, translesion synthesis by human DNA polymerase η was analyzed.     

Purification and characterization of a novel UV lesion-specific DNA glycosylase/AP lyase from Bacillus sphaericus

The purification and characterization of a pyrimidine dimer-specific glycosylase/AP lyase from Bacillus sphaericus (Bsp-pdg) are reported. Bsp-pdg is highly specific for DNA containing the cis-syn cyclobutane pyrimidine dimer, displaying no detectable activity on oligonucleotides with trans-syn I, trans-syn II, (6-4), or Dewar photoproducts. Like other glycosylase/AP lyases that sequentially cleave the N--glycosyl bond of the 5' pyrimidine of a cyclobutane pyrimidine dimer, and the phosphodiester backbone, this enzyme appears to utilize a primary amine as the attacking nucleophile. The formation of a covalent enzyme-DNA imino intermediate is evidenced by the ability to trap this protein-DNA complex by reduction with sodium borohydride. Also consistent with its AP lyase activity, Bsp-pdg was shown to incise an AP site-containing oligonucleotide, yielding beta- and delta-elimination products. N-terminal amino acid sequence analysis of this 26 kDa protein revealed little amino acid homology to any previously reported protein. This is the first report of a glycosylase/AP lyase enzyme from Bacillus sphaericus that is specific for cis-syn pyrimidine dimers.     

PCNA-induced DNA synthesis past cis-syn and trans-syn-I thymine dimers by calf thymus DNA polymerase delta in vitro.

Calf thymus proliferating cell nuclear antigen (PCNA) promoted DNA synthesis past cis-syn and trans-syn-I cyclobutane thymine dimers by calf thymus DNA polymerase delta (Pol delta) in vitro. Templates containing site-specific cis-syn and trans-syn-I thymine dimers were prepared via a combination of solid phase synthesis with photoproduct building blocks and DNA ligation. Extension of a 15-mer primer on the UV dimer-containing templates by Pol delta produced termination and bypass products in a dNTP and PCNA dependent manner. In the absence of PCNA and at dNTP concentrations varying between 1 and 100 microM, Pol delta could not bypass the cis-syn dimer and terminated elongation one nucleotide prior to the 3'-T of the dimer. DNA synthesis past the trans-syn-I dimer was even less efficient. In the presence of PCNA, termination occurred primarily one nucleotide prior to the 3'-T of both dimers at 1 microM dNTPs but opposite the 5'-T of the dimers at 100 microM dNTPs. In addition, under the latter conditions, bypass of the dimers was observed, to the extent of about 30% of the products for the cis-syn dimer and about 15% for the trans-syn-I dimer.     

Synthesis and nucleosome structure of DNA containing a UV photoproduct at a specific site.

A strategy was developed to assemble nucleosomes specifically damaged at only one site and one structural orientation. The most prevalent UV photoproduct, a cis-syn cyclobutane thymine dimer (cs CTD), was chemically synthesized and incorporated into a 30 base oligonucleotide harboring the glucocorticoid hormone response element. This oligonucleotide was assembled into a 165 base pair double stranded DNA molecule with nucleosome positioning elements on each side of the cs CTD-containing insert. Proton NMR verified that the synthetic photoproduct is the cis-syn stereoisomer of the CTD. Moreover, two different pyrimidine dimer-specific endonucleases cut approximately 90% of the dsDNA molecules. This cleavage is completely reversed by photoreactivation with E. coli UV photolyase, further demonstrating the correct stereochemistry of the photoproduct. Nucleosomes were reconstituted by histone octamer exchange from chicken erythocyte core particles, and contained a unique translational and rotational setting of the insert on the histone surface. Hydroxyl radical footprinting demonstrates that the minor groove at the cs CTD is positioned away from the histone surface about 5 bases from the nucleosome dyad. Competitive gel-shift analysis indicates there is a small increase in histone binding energy required for the damaged fragment (DeltaDeltaG approximately 0.15 kcal/mol), which does not prevent complete nucleosome loading under our conditions. Finally, folding of the synthetic DNA into nucleosomes dramatically inhibits cleavage at the cs CTD by T4 endonuclease V and photoreversal by UV photolyase. Thus, specifically damaged nucleosomes can be experimentally designed for in vitro DNA repair studies.     

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.     

Roles of the four DNA polymerases of the crenarchaeon Sulfolobus solfataricus and accessory proteins in DNA replication

The hyperthermophilic crenarchaeon Sulfolobus solfataricus P2 encodes three B-family DNA polymerase genes, B1 (Dpo1), B2 (Dpo2), and B3 (Dpo3), and one Y-family DNA polymerase gene, Dpo4, which are related to eukaryotic counterparts. Both mRNAs and proteins of all four DNA polymerases were constitutively expressed in all growth phases. Dpo2 and Dpo3 possessed very low DNA polymerase and 3' to 5' exonuclease activities in vitro. Steady-state kinetic efficiencies (k(cat)/K(m)) for correct nucleotide insertion by Dpo2 and Dpo3 were several orders of magnitude less than Dpo1 and Dpo4. Both the accessory proteins proliferating cell nuclear antigen and the clamp loader replication factor C facilitated DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis by Dpo2 and Dpo3 was remarkably decreased by single-stranded binding protein, in contrast to Dpo1 and Dpo4. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein was most processive with Dpo1, whereas DNA lesion bypass was most effective with Dpo4. Both Dpo2 and Dpo3, but not Dpo1, bypassed hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypassed uracil and cis-syn cyclobutane thymine dimer, respectively. High concentrations of Dpo2 or Dpo3 did not attenuate DNA synthesis by Dpo1 or Dpo4. We conclude that Dpo2 and Dpo3 are much less functional and more thermolabile than Dpo1 and Dpo4 in vitro but have bypass activities across hypoxanthine, 8-oxoguanine, and either uracil or cis-syn cyclobutane thymine dimer, suggesting their catalytically limited roles in translesion DNA synthesis past deaminated, oxidized base lesions and/or UV-induced damage.     

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)).     

Deamination of 5-methylcytosines within cyclobutane pyrimidine dimers is an important component of UVB mutagenesis

UVB mutagenesis is characterized by an abundance of C --> T and 5-methylcytosine --> T transitions at dipyrimidine sequences. It is not known how these mutations might arise. One hypothesis is that UV-induced mutations occur only after deamination of the cytosine or 5-methylcytosine within the pyrimidine dimer. It is not clear how methylation of cytosines at the 5-position influences deamination and how this affects mutagenesis. We have now conducted experiments with a CpG-methylated supF shuttle vector that was irradiated with UVB and then incubated at 37 degrees C to allow time for deamination before passage through a human cell line to establish mutations. This led to a significantly increased frequency of CC --> TT mutations and of transition mutations at 5'-PymCG-3' sequences. A spectrum of deaminated cis-syn cyclobutane pyrimidine dimers in the supF gene was determined using the mismatch glycosylase activities of MBD4 protein in combination with ligation-mediated PCR. Methylation at the C-5 position promoted the deamination of cytosines within cis-syn cyclobutane pyrimidine dimers, and these two events combined led to a significantly increased frequency of UVB-induced transition mutations at 5'-PymCG-3' sequences. Under these conditions, the majority of all supF mutations were transition mutations at 5'-PymCG-3', and they clustered at several mutational hot spots. Exactly these types of mutations are frequently observed in the p53 gene of nonmelanoma skin tumors. This particular mutagenic pathway may become prevalent under conditions of inefficient DNA repair and slow proliferation of cells in the human epidermis.     

The catalytic mechanism of a pyrimidine dimer-specific glycosylase (pdg)/abasic lyase, Chlorella virus-pdg

The repair of UV light-induced cyclobutane pyrimidine dimers can proceed via the base excision repair pathway, in which the initial step is catalyzed by DNA glycosylase/abasic (AP) lyases. The prototypical enzyme studied for this pathway is endonuclease V from the bacteriophage T4 (T4 bacteriophage pyrimidine dimer glycosylase (T4-pdg)). The first homologue for T4-pdg has been found in a strain of Chlorella virus (strain Paramecium bursaria Chlorella virus-1), which contains a gene that predicts an amino acid sequence homology of 41% with T4-pdg. Because both the structure and critical catalytic residues are known for T4-pdg, homology modeling of the Chlorella virus pyrimidine dimer glycosylase (cv-pdg) predicted that a conserved glutamic acid residue (Glu-23) would be important for catalysis at pyrimidine dimers and abasic sites. Site-directed mutations were constructed at Glu-23 to assess the necessity of a negatively charged residue at that position (Gln-23) and the importance of the length of the negatively charged side chain (Asp-23). E23Q lost glycosylase activity completely but retained low levels of AP lyase activity. In contrast, E23D retained near wild type glycosylase and AP lyase activities on cis-syn dimers but completely lost its activity on the trans-syn II dimer, which is very efficiently cleaved by the wild type cv-pdg. As has been shown for other glyscosylases, the wild type cv-pdg catalyzes the cleavage at dimers or AP sites via formation of an imino intermediate, as evidenced by the ability of the enzyme to be covalently trapped on substrate DNA when the reactions are carried out in the presence of a strong reducing agent; in contrast, E23D was very poorly trapped on cis-syn dimers but was readily trapped on DNA containing AP sites. It is proposed that Glu-23 protonates the sugar ring, so that the imino intermediate can be formed.     

T4 DNA polymerase (3''-5'') exonuclease, an enzyme for the detection and quantitation of stable DNA lesions: the ultraviolet light example.

Ultraviolet light irradiation of DNA results in the formation of two major types of photoproducts, cyclobutane dimers and 6-4' [pyrimidin-2'-one] -pyrimidine photoproducts. The enzyme T4 DNA polymerase possesses a 3' to 5' exonuclease activity and hydrolyzes both single and double stranded DNA in the absence of deoxynucleotide triphosphate substrates. Here we describe the use of T4 DNA polymerase associated exonuclease for the detection and quantitation of UV light-induced damage on both single and double stranded DNA. Hydrolysis of UV-irradiated single or double stranded DNA by the DNA polymerase associated exonuclease is quantitatively blocked by both cyclobutane dimers and (6-4) photoproducts. The enzyme terminates digestion of UV-irradiated DNA at the 3' pyrimidine of both cyclobutane dimers and (6-4) photoproducts. For a given photoproduct site, the induction of cyclobutane dimers was the same for both single and double stranded DNA. A similar relationship was also found for the induction of (6-4) photoproducts. These results suggest that the T4 DNA polymerase proofreading activity alone cannot remove these UV photoproducts present on DNA templates, but instead must function together with enzymes such as the T4 pyrimidine dimer-specific endonuclease in the repair of DNA photoproducts. The T4 DNA polymerase associated exonuclease should be useful for the analysis of a wide variety of bulky, stable DNA adducts.     

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.     

Characterization of distamycin A binding to damaged DNA

We previously reported that distamycin A, a natural antibiotic known as a minor groove binder, could bind to DNA duplexes containing the (6-4) photoproduct formed at its target site, whereas the binding was not observed for duplexes containing the cis-syn cyclobutane pyrimidine dimer in the same sequence context. In this study, we have further analyzed the binding of this drug to lesion-containing duplexes to elucidate its damaged-DNA recognition mechanism. Surface plasmon resonance measurements using various types of DNA showed that distamycin A could bind to several types of lesion-containing DNA. Curve fitting of the CD titration data revealed that the complex formation occurred with K(d) values around 10(-6) and a stoichiometry of 1:1. The results obtained in this study suggested that distamycin A binds to damaged DNA in the same way as to the normal target site, by recognizing the chemical structure of the minor groove.     

Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V

The ability of HeLa DNA polymerases to carry out DNA synthesis from incisions made by various endodeoxyribonucleases which recognize or form baseless sites in DNA was examined. DNA polymerase beta carried out limited strand displacement synthesis from 3'-hydroxyl nucleotide termini made by HeLa apurinic/apyrimidinic (AP) endonuclease II at the 5'-side of apurinic sites. Escherichia coli endonuclease III incises at the 3'-side of apurinic sites to produce nicks with 3'-deoxyribose termini which did not efficiently support DNA synthesis with beta-polymerase. However, these nicks could be activated to support limited DNA synthesis by HeLa AP endonuclease II, an enzyme which removes the baseless sugar phosphate from the 3'-termini, thus creating a one-nucleotide gap. With dGTP as the only nucleoside triphosphate present, the beta-polymerase catalyzed one-nucleotide DNA repair synthesis from those gaps which lacked dGMP. In contrast, HeLa DNA polymerase alpha was unreactive with all of the above incised DNA substrates. Larger patches of DNA synthesis were produced by nick translation from one-nucleotide gaps with HeLa DNA polymerase beta and HeLa DNase V. Moreover, incisions made by E. coli endonuclease III were activated to support DNA synthesis by the DNase V which removed the 3'-deoxyribose termini. HeLa DNase V also stimulated both the rate and extent of DNA synthesis by DNA polymerase beta from AP endonuclease II incisions. In this case the baseless sugar phosphate was removed from the 5'-termini, and nick translational synthesis occurred. Complete DNA excision repair of pyrimidine dimers was achieved with the beta-polymerase, DNase V, and DNA ligase from incisions made in UV-irradiated DNA by T4 UV endonuclease and HeLa AP endonuclease II. Such incisions produce a one-nucleotide gap containing 3'-hydroxyl nucleotide and 5'-thymine: thymidylate cyclobutane dimer termini. DNase V removes pyrimidine dimers primarily as a dinucleotide and then promotes nick translational DNA synthesis.     

Requirement of DNA polymerase eta for error-free bypass of UV-induced CC and TC photoproducts

The yeast RAD30-encoded DNA polymerase eta (Poleta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Human DNA polymerase eta functions similarly in the bypass of this lesion, and mutations in human Poleta result in the cancer prone syndrome, the variant form of xeroderma pigmentosum. UV light, however, also elicits the formation of cis-syn cyclobutane dimers and (6-4) photoproducts at 5'-CC-3' and 5'-TC-3' sites, and in both yeast and human DNA, UV-induced mutations occur primarily by 3' C to T transitions. Genetic studies presented here reveal a role for yeast Poleta in the error-free bypass of cyclobutane dimers and (6-4) photoproducts formed at CC and TC sites. Thus, by preventing UV mutagenesis at a wide spectrum of dipyrimidine sites, Poleta plays a pivotal role in minimizing the incidence of sunlight-induced skin cancers in humans.     

Chemical synthesis and translesion replication of a cis-syn cyclobutane thymine-uracil dimer

The cytosine base in DNA undergoes hydrolytic deamination at a considerable rate when UV radiation induces formation of a cyclobutane pyrimidine dimer (CPD) with an adjacent pyrimidine base. We have synthesized a phosphoramidite building block of a cis–syn cyclobutane thymine–uracil dimer (T[]U), which is the deaminated form of the CPD at a TC site, and incorporated it into oligodeoxyribonucleotides. The previously reported method for synthesis of the thymine dimer (T[]T) was applied, using partially protected thymidylyl-(3′–5′)-2′-deoxyuridine as the starting material, and after triplet- sensitized irradiation, the configuration of the base moiety in the major product was determined by NMR spectroscopy. Presence of the cis–syn cyclobutane dimer in the obtained oligonucleotides was confirmed by UV photoreversal and reaction with T4 endonuclease V. Using a 30mer containing T[]U, translesion synthesis by human DNA polymerase η was analyzed. There was no difference in the results between the templates containing T[]T and T[]U and pol η bypassed both lesions with the same efficiency, incorporating two adenylates. This enzyme showed fidelity to base pair formation, but this replication causes a C→T transition because the original sequence is TC.