Cis-syn Cyclobutane Thymine Dimers Research Articles

Identification of Three Human POLH Germline Variants Defective in Complementing the UV- and Cisplatin-Sensitivity of POLH-Deficient Cells.

DNA polymerase (pol) η is responsible for error-free translesion DNA synthesis (TLS) opposite ultraviolet light (UV)-induced cis-syn cyclobutane thymine dimers (CTDs) and cisplatin-induced intrastrand guanine crosslinks. POLH deficiency causes one form of the skin cancer-prone disease xeroderma pigmentosum variant (XPV) and cisplatin sensitivity, but the functional impacts of its germline variants remain unclear. We evaluated the functional properties of eight human POLH germline in silico-predicted deleterious missense variants, using biochemical and cell-based assays. In enzymatic assays, utilizing recombinant pol η (residues 1-432) proteins, the C34W, I147N, and R167Q variants showed 4- to 14-fold and 3- to 5-fold decreases in specificity constants (kcat/Km) for dATP insertion opposite the 3'-T and 5'-T of a CTD, respectively, compared to the wild-type, while the other variants displayed 2- to 4-fold increases. A CRISPR/Cas9-mediated POLH knockout increased the sensitivity of human embryonic kidney 293 cells to UV and cisplatin, which was fully reversed by ectopic expression of wild-type pol η, but not by that of an inactive (D115A/E116A) or either of two XPV-pathogenic (R93P and G263V) mutants. Ectopic expression of the C34W, I147N, and R167Q variants, unlike the other variants, did not rescue the UV- and cisplatin-sensitivity in POLH-knockout cells. Our results indicate that the C34W, I147N, and R167Q variants-substantially reduced in TLS activity-failed to rescue the UV- and cisplatin-sensitive phenotype of POLH-deficient cells, which also raises the possibility that such hypoactive germline POLH variants may increase the individual susceptibility to UV irradiation and cisplatin chemotherapy.

Paradigm change in mutagenesis: polymerase tautomeric models for targeted, delayed and untargeted ultraviolet mutagenesis during error plication of double stranded DNA, containing cis-syn cyclobutane thymine dimers or thymine in rare tautomeric forms

Paradigm change in mutagenesis: polymerase tautomeric models for targeted, delayed and untargeted ultraviolet mutagenesis during error plication of double stranded DNA, containing cis-syn cyclobutane thymine dimers or thymine in rare tautomeric forms

Detection of UV-induced cyclobutane pyrimidine dimers by near-infrared spectroscopy and aquaphotomics.

Ultraviolet (UV) radiation causes cellular DNA damage, among which cyclobutane pyrimidine dimers (CPDs) are responsible for a variety of genetic mutations. Although several approaches have been developed for detection of CPDs, conventional methods require time-consuming steps. Aquaphotomics, a new approach based on near-infrared spectroscopy (NIRS) and multivariate analysis that determines interactions between water and other components of the solution, has become an effective method for qualitative and quantitative parameters measurement in the solutions. NIR spectral patterns of UVC-irradiated and nonirradiated DNA solutions were evaluated using aquaphotomics for detection of UV-induced CPDs. Groups of UV-irradiated and nonirradiated DNA samples were classified (87.5% accuracy) by soft independent modeling of class analogy (SIMCA). A precise regression model calculated from NIR water spectral patterns based on UVC doses (r Val = 0.9457) and the concentration of cis-syn cyclobutane thymine dimers (cis-syn T<>Ts; r Val = 0.9993) was developed using partial least squares regression (PLSR), while taking advantage of water spectral patterns, particularly around 1400–1500 nm. Our results suggested that, in contrast to DNA, the formation of cis-syn T<>Ts increased the strongly hydrogen bonded water. Additionally, NIRS could qualitatively and quantitatively detect cis-syn T<>Ts in isolated DNA aqueous solutions upon UVC exposure.

A Polymerase – Tautomeric Model for Targeted Frameshift Mutations: Deletions Formation during Error-prone or SOS Replication of Double-stranded DNA Containing cis-syn Cyclobutane Thymine Dimers

Now it is still unclear how frameshift mutations arise at cyclobutane pyrimidine dimers. The author develops polymerase – tautomeric model of ultraviolet mutagenesis. The model is described that is based on the formation of rare tautomeric bases in cis-syn cyclobutane thymine dimers. A mechanism was proposed for targeted deletions caused by cis-syn cyclobutane thymine dimers. Targeted deletions are frameshift mutations when one or several nucleotides are dropped out in a DNA site opposite to a lesion capable of stopping DNA synthesis. Ultraviolet irradiation may result in changes of tautomer states of DNA bases. Thymine molecule may form 5 rare tautomer forms. They are stable if these bases are part of cyclobutane dimers. Structural analysis indicates that opposite one type of cis-syn cyclobutane thymine dimers containing a single tautomeric base (TT2*, with the ‘*’ indicating a rare tautomeric base and the subscript referring to the particular conformation) it is impossible to insert any canonical DNA bases with the template bases with hydrogen bonds formation. Therefore it is proposed that under synthesis DNA containing cis-syn cyclobutane thymine dimers TT2* specialize or modified DNA polymerases will leave one nucleotide gaps opposite these cis-syn cyclobutane thymine dimers. Daughter DNA strand opposite cis-syn cyclobutane thymine dimers TT2* may fall out. If in opposite DNA strand the loop is formed, daughter strand becomes shorter. Some DNA nucleotides are lost. Targeted deletion is formed. According to the polymerase-tautomeric model of ultraviolet mutagenesis cis-syn cyclobutane thymine dimers wherein a thymine is in the canonical tautomeric forms do not result in mutations. Cis-syn cyclobutane thymine dimers wherein a thymine is in the rare tautomeric forms T1*, T4*, or T5* were shown to cause only targeted base substitution mutations. Cis-syn cyclobutane thymine dimers wherein a thymine is in the rare tautomeric form T2* may result in targeted frameshift mutations (targeted insertions and targeted deletions).

Mechanisms of targeted frameshift mutations: Insertions arising during error-prone or SOS synthesis of DNA containing cis-syn cyclobutane thymine dimers

It is still unclear how frameshift mutations arise at cyclobutane pyrimidine dimers. The polymerase model is commonly used to explain the mechanisms of various mutations. An alternative polymerase-tautomer model was developed for UV-induced mutagenesis. A mechanism was proposed for targeted insertions caused by cis-syn cyclobutane thymine dimers. Targeted insertions are frameshift mutations due to addition of one or more nucleotides in a DNA sequence opposite to a lesion capable of stopping DNA synthesis. Among other factors, cyclobutane pyrimidine dimers can cause targeted insertions. UV irradiation can change the tautomeric form of DNA bases. Five rare tautomeric forms are possible for thymine, and they are stable when the thymine is a component of a cyclobutane dimer. A structural analysis showed that none of the canonical nucleotides can be added opposite to a specific rare thymine tautomer so that hydrogen bonds form between the two bases. A single nucleotide gap is consequently left in the corresponding site of the nascent strand when a specialized or modified DNA polymerase drives SOS or error-prone DNA synthesis on a template containing cis-syn cyclobutane thymine dimers with a base occurring in the rare tautomeric form. If the DNA composition is homogenous within the region, the end of the growing DNA strand may slip to form a complementary pair with the nucleotide adjacent to the dimer according to the Streisinger model, thus producing a loop. A targeted insertion is thereby generated to make the daughter strand longer. Targeted insertions were for the first time assumed to result from the cis-syn cyclobutane thymine dimers wherein one or both of the bases occur in the specific tautomeric form that does not allow the addition and hydrogen bonding of any canonical nucleotide in the opposite position. A model was developed to explain how targeted insertions of one or more nucleotides are caused by cis-syn cyclobutane thymine dimers. Thus, the polymerase-tautomer model can explain the nature and formation of targeted frameshift mutations in addition to hot and cold spots or targeted or untargeted nucleotide substitutions.

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.

A Versatile DNA Nanochip for Direct Analysis of DNA Base‐Excision Repair

Direct observation of enzymes interacting with DNA should be one of the ultimate technologies for investigating the mechanical behavior of the enzymes during the reactions. Atomic force microscopy (AFM) enables observation of biomolecules at a nanoscale spatial resolution; however, for a stable analysis, a scaffold to observe the reaction should be explored. DNA origami has recently been developed for the construction of a wide variety of multidimensional nanostructures, which can be used as scaffolds to incorporate various functionalities at specific positions. We intended to construct an AFM-based analysis system for DNA repair using a DNA origami scaffold carrying various substrate double-stranded DNAs (dsDNA). We employed DNA baseexcision repair (BER) enzymes 8-oxoguanine glycosylase (hOgg1) and T4 pyrimidine dimer glycosylase (PDG) to analyze the reaction on the DNA scaffold (Figure 1a). In the repair process, hOgg1 removes 8-oxoguanine (oxoG) to prevent G:C!T:A transversion during replication by DNA glycosylase activity and endonuclease activity for apurine– apyrimidine (AP) sites (AP-lyase activity) (Figure 1a). PDG removes photodamaged pyrimidine dimer including cis–syn cyclobutane thymine dimer (T T) by DNA glycosylase/AP-lyase activity. BER enzymes often require structural changes of the target DNA strands, such as DNA bending, for the reaction to proceed. The enzyme hOgg1 bends double-helix DNA by about 708 with flipping out of the oxoG for procession of the excision. The enzyme PDG bends double-helix DNA by 608 with flipping out of the 3’-side of A in the opposite strand of T T. The glycosylase/AP-lyase activity of these enzymes leads to single-strand scission at the damaged nucleotide (Figure 1b). In addition, the reaction intermediates of both reactions form a covalent bond with the enzyme by reduction with NaBH4. [5,10] We recently developed a framelike DNA origami scaffold to incorporate two different substrate dsDNAs. If the above-mentioned enzymatic and chemical reactions

Structure and mechanism of human DNA polymerase η

The variant form of human xeroderma pigmentosum syndrome (XPV) is caused by a deficiency in DNA polymerase η (Pol η) that enables replication through sunlight-induced pyrimidine dimers. We report high-resolution crystal structures of human Pol η at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol η acts like a molecular splint to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol η orthologs form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. Based on the structures, eight Pol η missense mutations causing XPV can be rationalized as undermining the “molecular splint” or perturbing the active-site alignment. The structures also shed light on the role of Pol η in replicating through D loop and DNA fragile sites.

Role of the ubiquitin-binding domain of Polη in Rad18-independent translesion DNA synthesis in human cell extracts

In eukaryotic cells, the Rad6/Rad18-dependent monoubiquitination of the proliferating cell nuclear antigen (PCNA) plays an essential role in the switching between replication and translesion DNA synthesis (TLS). The DNA polymerase Polη binds to PCNA via a consensus C-terminal PCNA-interacting protein (PIP) motif. It also specifically interacts with monoubiquitinated PCNA thanks to a recently identified ubiquitin-binding domain (UBZ). To investigate whether the TLS activity of Polη is always coupled to PCNA monoubiquitination, we monitor the ability of cell-free extracts to perform DNA synthesis across different types of lesions. We observe that a cis-syn cyclobutane thymine dimer (TT-CPD), but not a N-2-acetylaminofluorene-guanine (G-AAF) adduct, is efficiently bypassed in extracts from Rad18-deficient cells, thus demonstrating the existence of a Polη-dependent and Rad18-independent TLS pathway. In addition, by complementing Polη-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Polη activity. The finding that the bypass of a CPD lesion in vitro does not require Ub-PCNA but nevertheless depends on the UBZ domain of Polη, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA.

Ubiquitylation of Proliferating Cell Nuclear Antigen and Recruitment of Human DNA Polymerase η

This study investigated the requirement for ubiquitylation of PCNA at lysine 164 during polymerase eta-dependent translesion synthesis (TLS) of site-specific cis-syn cyclobutane thymine dimers (T (wedge)T). The in vitro assay recapitulated origin-dependent initiation, fork assembly, and semiconservative, bidirectional replication of double-stranded circular DNA substrates. A phosphocellulose column was used to fractionate HeLa cell extracts into two fractions; flow-through column fraction I (CFI) contained endogenous PCNA, RPA, ubiquitin-activating enzyme E1, and ubiquitin conjugase Rad6, and eluted column fraction II (CFII) included pol delta, pol eta, and RFC. CFII supplemented with purified recombinant RPA and PCNA (wild type or K164R, in which lysine was replaced with arginine) was competent for DNA replication and TLS. K164R-PCNA complemented CFII for these activities to the same extent and efficiency as wild-type PCNA. CFII mixed with CFI (endogenous PCNA, E1, Rad6) exhibited enhanced DNA replication activity, but the same TLS efficiency determined with the purified proteins. These results demonstrate that PCNA ubiquitylation at K164 of PCNA is not required in vitro for pol eta to gain access to replication complexes at forks stalled by T (wedge)T and to catalyze TLS across this dimer.

50 years thymine dimer

Fifty years ago thymine dimer was discovered in the Biochemical and Biophysical Laboratory of Delft Technological University, The Netherlands, by one of the authors of this review (Beukers) as the first environmentally induced DNA lesion. It is one of the photoproducts formed between adjacent pyrimidine bases in DNA by UV irradiation, currently known as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Major lesions found in DNA after in vitro or in vivo UV irradiation are the cis–syn cyclobutane thymine dimer and the thymine–cytosine (6-4) photoproduct. Even after 50 years the study of photo-induced DNA lesions is still going on as is illustrated by the hundreds of papers published every year and the millions hits when browsing the internet for dimer-related information. Living organisms possess efficient and different mechanisms to repair detrimental lesions in their DNA. A unique mechanism to repair CPDs is reversion by either direct interaction with light of short wavelength or by enzymatic photoreactivation. Photophysical mechanisms that induce and reverse molecular bonds in biological macromolecules have been a main focus of research of the group in Delft in the middle of the last century. This review describes the break-through results of these studies which were the result of intense interactions between scientists in the fields of physics, organic chemistry and biochemistry. Philosophically, the “view” of the group in Delft was very appealing: since in nature photolesions are induced in DNA by the sun, how is it possible that repair of these lesions could be accomplished by the same energy source. Evolutionary, it is hardly possible to think of a more efficient repair mechanism.

A human DNA polymerase η complex containing Rad18, Rad6 and Rev1; proteomic analysis and targeting of the complex to the chromatin‐bound fraction of cells undergoing replication fork arrest

DNA polymerase eta (Poleta) is responsible for efficient translesion synthesis (TLS) past cis-syn cyclobutane thymine dimers (TT dimers), the major DNA lesions induced by UV irradiation. Loss of human Poleta leads to xeroderma pigmentosum variant syndrome, clearly indicating that Poleta plays a vital role in preventing skin cancer caused by exposure to sunlight. To further examine Poleta functions and the mechanisms that regulate this important protein, Poleta complexes were purified from HeLa cells over-expressing epitope-tagged Poleta, and polypeptides associated with Poleta, including Rad18, Rad6 and Rev1, were identified by a combination of mass spectrometry and Western blot analysis. The chromatin-bound fractions of cells subjected to UV irradiation, S phase synchronization, or S phase arrest were specifically enriched in such complexes. These results suggest that arrested replication forks strengthen interactions among Poleta, Rad18/Rad6 and Rev1, consistent with the requirement for effective TLS by Poleta at sites of DNA lesions.

DNA binding properties of human DNA polymerase η: implications for fidelity and polymerase switching of translesion synthesis

The human XPV (xeroderma pigmentosum variant) gene is responsible for the cancer-prone xeroderma pigmentosum syndrome and encodes DNA polymerase eta (pol eta), which catalyses efficient translesion synthesis past cis-syn cyclobutane thymine dimers (TT dimers) and other lesions. The fidelity of DNA synthesis by pol eta on undamaged templates is extremely low, suggesting that pol eta activity must be restricted to damaged sites on DNA. Little is known, however, about how the activity of pol eta is targeted and restricted to damaged DNA. Here we show that pol eta binds template/primer DNAs regardless of the presence of TT dimers. Rather, enhanced binding to template/primer DNAs containing TT dimers is only observed when the 3'-end of the primer is an adenosine residue situated opposite the lesion. When two nucleotides have been incorporated into the primer beyond the TT dimer position, the pol eta-template/primer DNA complex is destabilized, allowing DNA synthesis by DNA polymerases alpha or delta to resume. Our study provides mechanistic explanations for polymerase switching at TT dimer sites.

Binding of Zinc Finger Protein Transcription Factor IIIA to Its Cognate DNA Sequence with Single UV Photoproducts at Specific Sites and Its Effect on DNA Repair

The relationship between DNA repair efficiency at specific locations in the binding site of the nine-zinc finger protein transcription factor IIIA (TFIIIA) and binding of its individual zinc fingers was studied. Homogeneously damaged oligonucleotides, which contained a single cis-syn cyclobutane thymine dimer (CTD) at one of six different sites in the internal control region (ICR) of the 5 S rRNA gene to generate a series of damaged DNA substrates, were prepared by chemical synthesis. Binding of TFIIIA to the substrates was assayed by measurement of dissociation constants (Kd), dissociation rates (koff), and protein-DNA contacts. The results indicated that a single CTD in the ICR does not significantly affect the Kd of TFIIIA. In contrast, CTDs at positions +55 and +72 (from the transcription start site) in the ICR markedly enhanced koff of TFIIIA from the complex. In addition, CTDs in these two sites increased methylation of the N7 of guanines (by dimethyl sulfate) in the zinc finger contacts of the ICR-TFIIIA complex. Furthermore CTDs at +55 and +72 were more efficiently removed from the complex than CTDs at other sites in the ICR by Xenopus oocyte nuclear extracts. This suggests that repair of CTDs closely correlates with changes in the binding of individual zinc fingers of the ICR-TFIIIA complex. These results have implications for the mechanism of DNA damage recognition and repair in protein-DNA complexes.

Mutations in human DNA polymerase eta motif II alter bypass of DNA lesions.

Human DNA polymerase eta (hPol eta) is one of the newly identified Y-family of DNA polymerases. These polymerases synthesize past template lesions that are postulated to block replication fork progression. hPol eta accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and contributes to normal resistance to sunlight-induced skin cancer. We describe here mutational analysis of motif II, a highly conserved sequence, recently reported to reside in the fingers domain and to form part of the active site in Y-family DNA polymerases. We used a yeast-based complementation system to isolate biologically active mutants created by random sequence mutagenesis, synthesized the mutant proteins in vitro and assessed their ability to bypass thymine dimers. The mutability of motif II in 210 active mutants has parallels with natural evolution and identifies Tyr52 and Ala54 as prime candidates for involvement in catalytic activity or bypass. We describe the ability of hPol eta S62G, a mutant polymerase with enhanced activity, to bypass five other site-specific lesions. Our results may serve as a prototype for studying other members of the Y-family DNA polymerases.

Formation of the Main UV-induced Thymine Dimeric Lesions within Isolated and Cellular DNA as Measured by High Performance Liquid Chromatography-Tandem Mass Spectrometry

UVB radiation-induced formation of dimeric photoproducts at bipyrimidine sites within DNA has been unambiguously associated with the lethal and mutagenic properties of sunlight. The main lesions include the cyclobutane pyrimidine dimers and the pyrimidine (6-4) pyrimidone adducts. The latter compounds have been shown in model systems to be converted into their Dewar valence isomers upon exposure to UVB light. A new direct assay, based on the use of liquid chromatography coupled to tandem mass spectrometry, is now available to simultaneously detect each of the thymine photoproducts. It was applied to the determination of the yields of formation of the thymine lesions within both isolated and cellular DNA exposed to either UVC or UVB radiation. The cis-syn cyclobutane thymine dimer was found to be the major photoproduct within cellular DNA, whereas the related (6-4) adduct was produced in an approximately 8-fold lower yield. Interestingly, the corresponding Dewar valence isomer could not be detected upon exposure of human cells to biologically relevant doses of UVB radiation.

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.

Solution-state structure of a DNA dodecamer duplex containing a Cis-Syn thymine cyclobutane dimer, the major UV photoproduct of DNA

The solution structures of a duplex DNA dodecamer containing a cis-syn cyclobutane thymine dimer d(GCACGAAT[ cs]TAAG)·d(CTTAATTCG TGC) and its native parent sequence were determined using NMR data collected at 750 MHz. The dodecamer sequence corresponds to the section of a site-specific cis-syn dimer containing 49-mer that was found to be the binding site for the dimer-specific T4 denV endonuclease V repair enzyme by chemical and enzymatic footprinting experiments. Structures of both sequences were derived from NOE restrained molecular dynamics/simulated annealing calculations using a fixed outer layer of water and an inner dynamic layer of water with sodium counterions. The resulting structures reveal a subtle distortion to the phosphodiester backbone in the dimer-containing sequence which includes a B II phosphate at the T9pA10 junction immediately 3′ to the dimer. The B II phosphate, established experimentally by analysis of the 31P chemical shifts and interpretation of the 3 J P-H3′ values using an optimized Karplus relationship, enables the DNA helix to accommodate the dimer by destacking the base 3′ to the dimer. Furthermore, the structures provide explanations for the unusually shifted T8-N3H imino, A16-H2 and T8-Me proton resonances and T9pA10 31P NMR resonance and are consistent with bending, unwinding, and thermodynamic data. The implications of the structural data for the mechanism by which cis-syn dimers are recognized by repair enzymes and bypassed by DNA polymerases are also discussed.

Specificities and Rates of Binding of Anti-(6-4) Photoproduct Antibody Fragments to Synthetic Thymine Photoproducts

Pyrimidine (6-4) pyrimidone photoproducts are some of the major DNA photolesions induced by ultraviolet (UV) light. A monoclonal antibody (64M5) specific to a (6-4) photoproduct has been established and the corresponding single-chain antibody (64M5scFv) has been prepared. In this study, we characterized the ligand selectivities of 64M5 and 64M5scFv using synthetic octadeoxynucleotides containing either a central cis-syn cyclobutane thymine dimer (T[c,s]T), the (6-4) photoproduct of TpT (T[6-4]T), or its Dewar isomer (T[Dewar]T) by means of enzyme-linked immunosorbent assays (ELISA). Both 64M5 and 64M5scFv recognized T[6-4]T, but not the other photoproducts. We synthesized several biotinylated oligonucleotides of different lengths containing (T[6-4]T) to analyze the effects of the antigen size on the binding rates of an antigen binding fragment (64M5Fab) and 64M5scFv by means of surface plasmon resonance. The association rate constants for oligonucleotides of different sizes containing T[6-4]T as to 64M5Fab were found to be almost the same (1.9-5.6 x 10(5) M(-1) x s(-1)), while the dissociation rate constant for the largest oligonucleotide (d8mer, 8.0 x 10(-5) s(-1)) was significantly smaller than that for the d2mer (4.2 x 10(-2) s(-1)). These results indicate that 64M5Fab recognized the d2mer as the epitope and that the binding affinity for T[6-4]T depended on the flanking oligonucleotides. The dissociation rate constants for 64M5scFv as to the antigen analogs were almost the same as those for the various T[6-4]T-oligonucleotides as to 64M5Fab, suggesting that the conformations of these antibody binding regions are pretty similar to each other.

Taq DNA polymerase blockage at pyrimidine dimers

The most stable DNA-lesions generated by irradiation with ultraviolet light (254 nm) are two kinds of pyrimidine dimers (PDs), cylobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PP) (1). Blockage of DNA-polymerase from Thermus aquaticus (Taq polymerase) at PDs has been applied in primer extension assays for photofootprinting (2) and for mapping PDs at nucleotide resolution (3,4). These assays depend on an efficient and precise blockage at PDs. Using plasmid DNA with a site specific CPD or 6-4PP and irradiated yeast DNA, we report here that Taq DNA polymerase is indeed completely blocked at PDs. Furthermore, we show that the signal depends on the local sequence and on whether the DNA was pretreated with T4-endonuclease V (T4-endoV) or with T4-endoV and DNAphotolyase. These results justify the use of primer extension protocols for quantitation of PDs and they point out some technical limitations of this approach. M13mp18TT-CPD is plasmid DNA containing a unique cis–syn cyclobutane thymine dimer (5). The DNA was cut with HindIII and aliquots were treated with (i) T4-endoV, which cuts at the CPD, (ii) DNA-photolyase, which reverts CPDs or (iii) T4-endoV and then DNA-photolyase. The DNA samples were denatured, an oligonucleotide labeled at the 5′-end with 32P was hybridized and primer extension by Taq DNA polymerase was done for 30 cycles. The reaction products were displayed on a sequencing gel and compared with sequencing lanes obtained by dideoxy sequencing using the same primer extension protocol (Fig. 1a). With untreated DNA, 93 ± 5% of the signal (average of three experiments) accumulated in front of or at the CPD (lane 5, region B) and 7% was detected at the HindIII site (region A). Pretreatment with T4-endoV (lane 6) or T4-endoV and photolyase (lanes 7) changed the signal in the B and A regions by <2% in individual experiments (see legend to Fig. 1). This result shows that blockage at the CPD site was complete and that the signal at the HindIII site corresponds to a fraction of plasmid DNA without a CPD and was not due to read through at the CPD site. After photolyase treatment, all signal was found at the HindIII site (lane 8), demonstrating that blockage in lanes 5–7 was indeed due to a CPD. Blockage at the CPD results in two major bands migrating at the position of the 3′T and 3′G, and a few minor bands which indicate premature stops (lane 5, region B). T4-endoV cleavage results in a major band at the 3′T and a minor band at th 5′T (lane 6). Additional removal of the overhanging 5′T by photolyase results in a minor band at the 3′T and major band at the site of the removed 5′T (lane 7). Since Taq polymerase is known to frequently add a non-template 3′ adenosine residue (6), the results Figure 1. Blockage of Taq DNA polymerase at a CPD (a) and 6-4PP (b). (a) Primer extension is shown for M13mp18TT-CPD DNA cut with HindIII (lane 5), and treated with T4-endoV (lane 6), T4-endoV then photolyase (lane 7) or photolyase only (lane 8). Dideoxy sequencing lanes are also shown (lanes 1–4). The relevant part of the sequence is shown (5′-AGTTGGAGC-3′). Boxes A indicate elongation to the HindIII cut. Boxes B represent blockage in the thymine dimer region. The signals in region B were almost constant in lanes 5, 6 and 7: 95, 95 and 96% (experiment 1); 98, 99 and 96% (experiment 2); 88, 86 and 89% (experiment 3; the values were lower than in experiments 1 and 2 due to higher gel background). Region A plus region B is 100%. (b) Primer extension is shown for M13mp18TT-64 DNA, cut with HindIII (lane 5) and treated with piperidine (lane 6). The sequence is shown in lanes 1–4. The signals in region B of untreated samples (lanes 5) were 84 ± 1% and for piperidine treated samples 78 ± 10% (average of three experiments).