DNA polymerase iota-dependent translesion replication of uracil containing cyclobutane pyrimidine dimers

Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type S-phase cells reveals that only approximately 25% of mutations occur at thymine (T), whilst 75% are targeted to cytosine (C). The mutational spectra changes dramatically in XP-V cells, devoid of poleta, where approximately 45% of mutations occur at Ts and approximately 55% at Cs. At the present time, it is unclear whether the C-->T mutations actually represent true misincorporations opposite C, or perhaps occur as the result of the correct incorporation of adenine (A) opposite a C in a UV-photoproduct that had undergone deamination to uracil (U). In order to assess the role that human poliota might play, if any, in the replicative bypass of such UV-photoproducts, we have analyzed the efficiency and fidelity of pol iota-dependent bypass of a T-U cyclobutane pyrimidine dimer (CPD) in vitro. Interestingly, pol iota-dependent bypass of a T-U CPD occurs more efficiently than that of a corresponding T-T CPD. Guanine (G) was misincorporated opposite the 3'U of the T-U CPD only two-fold less frequently than the correct Watson-Crick base, A. While pol iota generally extended the G:3'U-CPD mispairs less efficiently than the correctly paired primer, pol iota-dependent extension was equal to, or greater than that observed with human pols eta and kappa and S. cerevisiae pol zeta under the same assay conditions. Thus, we hypothesize that the ability of pol iota to bypass T-U CPDs through the frequent misincorporation of G opposite the 3'U of the CPD, may provide a mechanism whereby human cells can decrease the mutagenic potential of these lesions.     

In vivo recruitment of XPC to UV-induced cyclobutane pyrimidine dimers by the DDB2 gene product

The initial step in mammalian nucleotide excision repair (NER) of the major UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs), requires lesion recognition. It is believed that the heterodimeric proteins XPC/hHR23B and UV-DDB (UV-damaged DNA binding factor, composed of the p48 and p127 subunits) perform this function in genomic DNA, but their requirement and lesion specificity in vivo remains unknown. Using repair-deficient xeroderma pigmentosum (XP)-A cells that stably express photoproduct-specific photolyases, we determined the binding characteristics of p48 and XPC to either CPDs or 6-4PPs in vivo. p48 localized to UV-irradiated sites that contained either CPDs or 6-4PPs. However, XPC localized only to UV-irradiated sites that contained 6-4PPs, suggesting that XPC does not efficiently recognize CPDs in vivo. XPC did localize to CPDs when p48 was overexpressed in the same cell, signifying that p48 activates the recruitment of XPC to CPDs and may be the initial recognition factor in the NER pathway.     

Physical and functional interaction between DDB and XPA in nucleotide excision repair

Damaged DNA-binding protein (DDB), consisting of DDB1 and DDB2 subunits recognizes a wide spectrum of DNA lesions. DDB is dispensable for in vitro nucleotide excision repair (NER) reaction, but stimulates this reaction especially for cyclobutane pyrimidine dimer (CPD). Here we show that DDB directly interacts with XPA, one of core NER factors, mainly through DDB2 subunit and the amino-acid residues between 185 and 226 in XPA are important for the interaction. Interestingly, the point mutation causing the substitution from Arg-207 to Gly, which was previously identified in a XP-A revertant cell-line XP129, diminished the interaction with DDB in vitro and in vivo. In a defined system containing R207G mutant XPA and other core NER factors, DDB failed to stimulate the excision of CPD, although the mutant XPA was competent for the basal NER reaction. Moreover, in vivo experiments revealed that the mutant XPA is recruited to damaged DNA sites with much less efficiency compared with wild-type XPA and fails to support the enhancement of CPD repair by ectopic expression of DDB2 in SV40-transformed human cells. These results suggest that the physical interaction between DDB and XPA plays an important role in the DDB-mediated NER reaction.     

Early steps of excision repair of cyclobutane pyrimidine dimers by the Micrococcus luteus endonuclease. A three-step incision model

The early steps of excision repair of cyclobutane pyrimidine dimers are investigated. It is demonstrated that the apurinic/apyrimidinic endonuclease associated with the Micrococcus luteus uv-specific endonuclease cleaves the phosphodiester bond on the 3' side of the deoxyribose leaving a 3' hydroxy terminus and a 5' phosphoryl terminus. This nick is not a substrate for T4 polynucleotide ligase. The 3' base-free deoxyribose terminus is not a substrate for either the polymerase or the 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I. However, the 3' terminus of the nick is converted to a substrate for DNA polymerization by the action of a 5' apurinic/apyrimidinic endonuclease. A three-step model for the incision step of excision repair of cyclobutane pyrimidine dimers is presented.     

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.     

Immunoprecipitation of pyrimidine(6-4)pyrimidone photoproducts and cyclobutane pyrimidine dimers in uv-irradiated DNA

Biological studies suggest that a significant proportion of the cytotoxicity observed in mammalian cells after uv irradiation may be due to damage other than cyclobutane dimers in DNA. Although pyrimidine-pyrimidone (6-4) photoproducts have been implicated as major contributors to cell lethality, their induction has been measured at considerably less than cyclobutane pyrimidine dimers when measured by chromatographic techniques. Because the yield of (6-4) photoproducts may be reduced by their lability to extreme heat and pH, we have advised an alternative, immunological quantification which does not require DNA hydrolysis. Affinity-purified rabbit antisera were used to precipitate low molecular weight 32P-labeled PM2 DNA irradiated with increasing fluences of uv light. DNA of known molecular weight was used to determine rates of induction for antibody-binding sites associated with (6-4) photoproducts and cyclobutane dimers. These rates were calculated to be 0.6 (6-4) photoproducts and 1.2 cyclobutane dimers/10(8) Da/J/m2. At low uv fluences (6-4) photoproducts were induced at one-half the rate of cyclobutane dimers, whereas at higher fluences (6-4) photoproducts predominated.     

Rapid immunoassays for detection of UV-induced cyclobutane pyrimidine dimers in whole bacterial cells

Immunoassays were developed to measure DNA damage retained by UV-irradiated whole bacterial cells. Active Mycobacterium parafortuitum and Serratia marcescens cells were fixed and incubated with cyclobutane pyrimidine dimer-binding antibodies after being exposed to known UV doses (254 nm). When both fluorescent (Alexa Fluor 488) and radiolabeled ((125)I) secondary antibodies were used as reporters, indirect whole-cell assays were sensitive enough to measure intracellular UV photoproducts in M. parafortuitum and S. marcescens cells as well as photoenzymatic repair responses in S. marcescens cells. For the same UV dose, fluorescent DNA photoproduct detection limits in whole-cell assays (immunofluorescent microscopy) were similar to those in fluorescent assays performed on membrane-bound DNA extracts (immunoslot blot). With either fluorescent or radiolabeled reporters, the intracellular cyclobutane pyrimidine dimer content of UV-irradiated whole bacterial cells could be reliably quantified after undergoing a <0.5-order-of-magnitude decrease in culturability. Immunofluorescent microscopy results showed that photoenzymatic repair competence is not uniformly distributed among exponential-growth UV-irradiated pure cultures.     

Enhanced repair of cyclobutane pyrimidine dimers and improved UV resistance in photolyase transgenic mice

During evolution, placental mammals appear to have lost cyclobutane pyrimidine dimer (CPD) photolyase, an enzyme that efficiently removes UV-induced CPDs from DNA in a light-dependent manner. As a consequence, they have to rely solely on the more complex, and for this lesion less efficient, nucleotide excision repair pathway. To assess the contribution of poor repair of CPDs to various biological effects of UV, we generated mice expressing a marsupial CPD photolyase transgene. Expression from the ubiquitous beta-actin promoter allowed rapid repair of CPDs in epidermis and dermis. UV-exposed cultured dermal fibroblasts from these mice displayed superior survival when treated with photoreactivating light. Moreover, photoreactivation of CPDs in intact skin dramatically reduced acute UV effects like erythema (sunburn), hyperplasia and apoptosis. Mice expressing the photolyase from keratin 14 promoter photo reactivate CPDs in basal and early differentiating keratinocytes only. Strikingly, in these animals, the anti-apoptotic effect appears to extend to other skin compartments, suggesting the presence of intercellular apoptotic signals. Thus, providing mice with CPD photolyase significantly improves repair and uncovers the biological effects of CPD lesions.     

An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.

Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. A second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential.     

Site-specific repair of cyclobutane pyrimidine dimers in a positioned nucleosome by photolyase and T4 endonuclease V in vitro.

Since genomic DNA is folded into nucleosomes, and DNA damage is generated all over the genome, a central question is how DNA repair enzymes access DNA lesions and how they cope with nucleosomes. To investigate this topic, we used a reconstituted nucleosome (HISAT nucleosome) as a substrate to generate DNA lesions by UV light (cyclobutane pyrimidine dimers, CPDs), and DNA photolyase and T4 endonuclease V (T4-endoV) as repair enzymes. The HISAT nucleosome is positioned precisely and contains a long polypyrimidine region which allows one to monitor formation and repair of CPDs over three helical turns. Repair by photolyase and T4-endoV was inefficient in nucleosomes compared with repair in naked DNA. However, both enzymes showed a pronounced site-specific modulation of repair on the nucleosome surface. Removal of the histone tails did not substantially enhance repair efficiency nor alter the site specificity of repair. Although photolyase and T4-endoV are different enzymes with different mechanisms, they exhibited a similar site specificity in nucleosomes. This implies that the nucleosome structure has a decisive role in DNA repair by exerting a strong constraint on damage accessibility. These findings may serve as a model for damage recognition and repair by more complex repair mechanisms in chromatin.     

UVA-induced cyclobutane pyrimidine dimers form predominantly at thymine-thymine dipyrimidines and correlate with the mutation spectrum in rodent cells

Ligation-mediated PCR was employed to quantify cyclobutane pyrimidine dimer (CPD) formation at nucleotide resolution along exon 2 of the adenine phosphoribosyltransferase (aprt) locus in Chinese hamster ovary (CHO) cells following irradiation with either UVA (340–400 nm), UVB (295–320 nm), UVC (254 nm) or simulated sunlight (SSL; λ > 295 nm). The resulting DNA damage spectrum for each wavelength region was then aligned with the corresponding mutational spectrum generated previously in the same genetic target. The DNA sequence specificities of CPD formation induced by UVC, UVB or SSL were very similar, i.e., in each case the overall relative proportion of this photoproduct forming at TT, TC, CT and CC sites was ~28, ~26, ~16 and ~30%, respectively. Furthermore, a clear correspondence was noted between the precise locations of CPD damage hotspots, and of ‘UV signature’ mutational hotspots consisting primarily of C→T and CC→TT transitions within pyrimidine runs. However, following UVA exposure, in strong contrast to the above situation for UVC, UVB or SSL, CPDs were generated much more frequently at TT sites than at TC, CT or CC sites (57% versus 18, 11 and 14%, respectively). This CPD deposition pattern correlates well with the strikingly high proportion of mutations recovered opposite TT dipyrimidines in UVA- irradiated CHO cells. Our results directly implicate the CPD as a major promutagenic DNA photoproduct induced specifically by UVA in rodent cells.     

Action spectra of DNA photolyases for photorepair of cyclobutane pyrimidine dimers in sorghum and cucumber

DNA photolyases that catalyze light-dependent repair of cyclobutane pyrimidine dimers (CPDs) were extracted and partially purified from sorghum and cucumber. The action spectra of CPD photolyases in these plant species had a maximum at 400 nm, which differ from those in Drosophila, Escherichia coli and Anacystis.     

Repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in the fission yeast Schizosaccharomyces pombe

We have measured repair of both of the major lesions induced by ultraviolet irradiation (cyclobutane pyrimidine dimers and 6-4 photoproducts) in wild-type Schizosaccharomyces pombe and in selected rad mutants, including mutants with deletions in genes from the main phenotypic groups. We find that rad13Δ, rad15 and rad16Δ, which are the S. pombe homologues of the excision-defective Saccharomyces cerevisiae rad2, rad3 and rad1, respectively, repair lesions somewhat more slowly than the wild type, but still have considerable repair capacity. rad2Δ, also a presumed excision-defective mutant, behaves similarly. radS and rad9δ, which belong to different phenotypic groups, repair lesions at the same rate as wild-type cells. These findings provide new evidence that S. pombe has a second repair system for removing ultraviolet damage, which is absent in S. cerevisiae. Surprisingly, this second mechanism repairs lesions very efficiently; its possible nature is discussed.     

Enhanced DNA repair of cyclobutane pyrimidine dimers changes the biological response to UV-B radiation

The goal of DNA repair enzyme therapy is the same as that for gene therapy: to rescue a defective proteome/genome by introducing a substitute protein/DNA. The danger of inadequate DNA repair is highlighted in the genetic disease xeroderma pigmentosum. These patients are hypersensitive to sunlight and develop multiple cutaneous neoplasms very early in life. The bacterial DNA repair enzyme T4 endonuclease V was shown over 25 years ago to be capable of reversing the defective repair in xeroderma pigmentosum cells. This enzyme, packaged in an engineered delivery vehicle, has been shown to traverse the stratum corneum, reach the nuclei of living cells of the skin, and enhance the repair of UV-induced cyclobutane pyrimidine dimers (CPD). In such a system, changes in DNA repair, mutagenesis, and cell signaling can be studied without manipulation of the genome.     

Sequence specificity of cyclobutane pyrimidine dimers in DNA treated with solar (ultraviolet B) radiation.

Cyclobutane pyrimidine dimers were quantified at the sequence level after irradiation with solar ultraviolet (UVB) and nonsolar ultraviolet (UVC) light sources. The yield of photoproducts at specific sites was dependent on the nucleotide composition in and around the potential lesion as well as on the wavelength of ultraviolet light used to induce the damage. Induction was greater in the presence of 5' flanking pyrimidines than purines; 5' guanine inhibited induction more than adenine. UVB irradiation increased the induction of cyclobutane dimers containing cytosine relative to thymine homodimers. At the single UVC and UVB fluences used, the ratio of thymine homodimers (T mean value of T) to dimers containing cytosine (C mean value of T, T mean value of C, C mean value of C) was greater after UVC compared to UVB irradiation.