Production and excision repair of cyclobutane-type pyrimidine dimers in the DNA of Turbatrix aceti

The production and removal of 254 nm ultraviolet-induced pyrimidine dimers was measured in the DNA of the free-living nematode Turbatrix aceti. Approximately 0.0035 per cent pyrimidine dimers are produced per J/m2. Following a fluence of 100 Jm2, approximately 50 per cent of the dimeric photoproducts were excised within 60 min. The number of pyrimidine dimers excised did not change with increasing U.V. fluence, indicating saturation of the U.V. repair system in T. aceti. The results indicate a highly efficient and selective repair system in Turbatrix aceti for dimeric photoproducts.     

Repair of DNA-containing pyrimidine dimers

Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.     

Bypass of pyrimidine dimers in DNA of bacteriophage T4 via induction of primer RNA

Bacteriophage T4 has a third pathway for repair of damaged DNA besides excision repair and recombination repair. This pathway is a mechanism for the toleration of lesions rather than the repair of lesions. The substrate for this process is gapped DNA copied from a damaged template. Evidence indicates that these gaps are filled, giving rise to daughter strands that are sensitive to heat and to treatments with RNAase. These daughter strands subsequently serve as templates for DNA that is resistant to RNAase. This third pathway is dependent upon gene 41 (RNA-priming protein), gene uvsZ (function unknown) and gene 30 (polynucleotide ligase) and is presumed to consist of 4 steps: (1) induction of primer RNA opposite the lesion in the template; (2) elongation of primers by DNA polymerase; (3) ligation of daughter-strand fragments, without removal of primer RNA; (4) replication of DNA carrying RNA sequences, giving homogeneous DNA strands. We have called this process 'Re-initiation repair'.     

Photomonomerization of pyrimidine dimers by indoles and proteins.

Model systems for the study of photoreactivation have been developed that utilize a variety of indole derivatives. These systems can split uracil cis-syn cyclobutadipyrimidine, either free or in RNA, when irradiated at wave-lengths absorbed only by the indole moiety. The ability of indole compounds to split dimers is closely related to their electronic properties. Those of high electron-donor capacity such as indole, 3-methylindole, indole-3-acetic acid, 5-hydroxytryptophan and tryptophan are good photosensitizers, with efficacy in that order. Indoles with electron-withdrawing substituents such as indole-3-carboxylic acid, indole-3-aldehyde and oxindole are inactive in the monomerization reaction. These findings support the proposed mechanism that the photosensitized monomerization occurs as a result of electron transfer from the excited indole molecules to the pyrimidine bases.Proteins containing fully exposed tryptophan residues (chicken egg white lysozyme and bovine diisopropylphosphoryltrypsin) also cause the splitting of the ¹⁴C-labeled dimers under the same conditions. In the case of lysozyme the quantum yield of monomerization is similar to that of free tryptophan. Much of the monomerization ability of lysozyme was lost after the solvent-available tryptophan had been oxidized by treatment with N-bromosuccinimide. Bovine pancreatic ribonuclease A, a protein devoid of tryptophan, failed to exhibit photosensitized monomerization of uracil dimers. The biological implication of these reactions involving a protein with an exposed tryptophan residue is discussed.Although indoles are able to split the dimers in RNA, they fail to photo-reactivate u.v.-damaged TMV-RNA. Indole-3-acetic acid, 3-methylindole and 5-hydroxytryptophan rapidly inactivate viral RNA when irradiated at 313 nm, possibly because of side reactions.     

Recognition in action: flipping pyrimidine dimers

DNA bases are normally sheltered within a double helix, but enzymes that modify and repair DNA gain access by flipping individual bases out of the double helix.     

Are pyrimidine dimers Non-Instructive lesions?

Summary Published data from yeast and E. coli show that base substitution induced by UV in pyrimidine-pyrimidine sequences is not random, and suggest that fidelity of DNA replication is not entirely lost during transdimer synthesis. These observations question whether cyclobutane pyrimidine dimers are truly non-instructive lesions.     

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.     

Inhibition of eukaryotic topoisomerase II by ultraviolet-induced cyclobutane pyrimidine dimers.

The effects of short wave ultraviolet (UV)-induced DNA lesions on the catalytic activity of Drosophila melanogaster topoisomerase II were investigated. The presence of these photoproducts impaired the enzyme's ability to relax negatively supercoiled pBR322 plasmid molecules. As determined by DNA photolyase-catalyzed photoreactivation experiments, enzyme inhibition was due to the presence of cyclobutane pyrimidine dimers in the DNA. When 10-20 cyclobutane dimers were present per plasmid, the initial velocity of topoisomerase II-catalyzed DNA relaxation was inhibited approximately 50%. Decreased relaxation activity correlated with an inhibition of the DNA strand passage step of the enzyme's catalytic cycle. In contrast, UV-induced photoproducts did not alter the prestrand passage DNA cleavage/religation equilibrium of topoisomerase II either in the absence or presence of antineoplastic agents. Results of the present study demonstrate that the repair of cyclobutane pyrimidine dimers is important for the efficient catalytic function of topoisomerase II.     

Photorepair of ultraviolet radiation-induced pyrimidine dimers in corneal DNA

The induction and photorepair of pyrimidine dimers in DNA have been measured in the ultraviolet-irradiated, corneal epithelium of the marsupial, Monodelphis domestica, using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that FS-40 sunlamps (280-400 nm) induced 7.2 +/- 1.0 X 10(-5) pyrimidine dimers per kilobase (kb) of DNA per J/m2. Following 100 J/m2, 50% and greater than 90% of the dimers were photorepaired during a 10- and 30-min exposure to photoreactivating light (320-400 nm), respectively. In addition, approximately 70% and approximately 60% of the dimers induced by 300 and 500 J/m2, respectively, were repaired by a 60-min exposure to photoreactivating light. The capacity of the corneal epithelium of M. domestica to photorepair pyrimidine dimers identifies this animal as a potentially useful model with which to determine whether pyrimidine dimers are involved in pathological changes of the irradiated eye.     

Excision of pyrimidine dimers in toluene-treated Escherichia coli.

Toluene-treated cells were used for examining excision of pyrimidine dimers in Escherichia coli strains W3110, DM845 (uvrA-), P3478 (polA-), and KS5064 (polAex1). Excision occurring in toluene-treated cells is rapid, adenosine 5'-triphosphate dependent, and requires the uvrA gene function. In strains lacking either the polymerizing or 5' leads to 3' exonucleolytic activity of deoxyribonucleic acid polymerase I, excision does occur. However, both in vivo and in vitro, the excision in such strains is initially slower than wild type.     

Retarded excision of pyrimidine dimers in human unstimulated lymphocytes

Using immuno-labelling of cyclobutane pyrimidine dimers (CPDs) in nuclei of peripheral lymphocytes after their UVC-irradiation and cultivation, we have found that within the first four hours of cultivation the CPD-specific fluorescent signal from cell nuclei increased. Earlier, a similar increase in binding of antibody specific for pyrimidine (6-4) pyrimidone photoproducts to undenatured DNA isolated from UV-irradiated Chinese hamster ovary cells was reported (Mitchell et al., 1986). Our experiments showed that nucleotide excision repair enzyme might induce such of DNA modification in lymphocyte nuclei that increased specific antibody binding to DNA fragments with lesions. We suggest that enzymatic formation of open structures in DNA predominated qualitatively over dual-incision and excision of these fragments, and resulted in the enhanced exposure of the pyrimidine dimers in nuclei to specific antibodies. The results evidence that nucleotid excision repair in unstimualted human lymphocytes being deficient in dual incision and removal of UV-induced DNA lesions appear to be capable of performing chromatin relaxation and pre-incision uncoiling of DNA fragments with lesions.     

The role of pyrimidine dimers in postreplication repair in Neurospora

Summary Using the Micrococcus luteus dimer specific endonuclease assay of Wilkins (1973), and photoreactivation we have examined the induction and fate of ultraviolet induced pyrimidine dimers in the excision defective strain, uvs-2, of Neurospora crassa.Dimer induction was fluence dependent from 0 to 800 ergs/mm² UV. An interdimer distance of 19.6x10⁶ DNA molecular weight was found after a fluence of 220 ergs/mm². We confirm the earlier report that this mutant is completely excision defective (Worthy and Epler 1972). Photoreactivation (PR), which greatly enhanced survival (by 10 fold after 440 ergs/mm² UV), reduced significantly (40–44%) the number of UV-endonuclease sensitive sites found in irradiated DNA. This treatment also alleviated immediately some of the temporary blocks to high molecular weight DNA synthesis (elongation or ligation) seen in irradiated cells.We have also attempted to elucidate the mechanism of cellular postreplication repair used to overcome the UV inhibition to DNA synthesis. It was determined that during postreplication repair, Neurospora does not use recombination to bypass dimers and that single stranded DNA gaps opposite dimers do not appear to be present during the time when DNA being synthesized is made only in short pieces.     

Excision of pyrimidine dimers by several UV-sensitive mutants of S. pombe

Summary Nine radiation-sensitive mutants of S. pombe showing a variety of phenotypic characteristics were analysed for their ability to excise pyrimidine dimers after ultraviolet irradiation. From earlier studies using indirect parameters, it was expected that some would be excision-deficient. Data reported here show that all the mutants tested, like wild type cells, were able to remove a high percentage of pyrimidine dimers during post-irradiation incubation in several different holding media, but not in saline or phosphate buffer. These mutants included strains showing increased, as well as others which showed decreased, levels of UV-induced mutation frequency relative to that of the wild type at the same total dose.     

Measurement of pyrimidine dimers in spheroplasts of Bacillus subtilis.

A method is described for making spheroplasts of Bacillus subtilis which are permeable to exogenous enzymes. Conditions are described for measuring small numbers of pyrimidine dimers in the DNA of UV-irradiated cells by use of a partially purified Micrococcus luteus extract containing an enzyme specific for pyrimidine dimers. The system will detect as few as 10-12 pyrimidine dimers per genome.