Utilization of DNA photolyase, pyrimidine dimer endonucleases, and alkali hydrolysis in the analysis of aberrant ABC excinuclease incisions adjacent to UV-induced DNA photoproducts.

ABC excinuclease of Escherichia coli removes 6-4 photoproducts and pyrimidine dimers from DNA by making two single strand incisions, one 8 phosphodiester bonds 5' and another 4 or 5 phosphodiester bonds 3' to the lesion. We describe in this communication a method, which utilizes DNA photolyase from E. coli, pyrimidine dimer endonucleases from M. luteus and bacteriophage T4, and alkali hydrolysis, for analyzing the ABC excinuclease incision pattern corresponding to each of these photoproducts in a DNA fragment. On occasion, ABC excinuclease does not incise DNA exclusively 8 phosphodiester bonds 5' or 4 or 5 phosphodiester bonds 3' to the photoproduct. Both the nature of the adduct (6-4 photoproduct or pyrimidine dimer) and the sequence of neighboring nucleotides influence the incision pattern of ABC excinuclease. We show directly that photolyase stimulates the removal of pyrimidine dimers (but not 6-4 photoproducts) by the excinuclease. Also, photolyase does not repair CC pyrimidine dimers efficiently while it does repair TT or TC pyrimidine dimers.     

Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase.

Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] are the two major photoproducts produced in DNA by UV irradiation. Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts [(6-4)photolyase]. (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism. The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family. Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity. Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD. This is the first indication of FAD as the chromophore of (6-4)photolyase.     

UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells

DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions. Immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as the immediate repair of CPDs, indicating that a single 6-4PP lesion is severalfold more toxic than a CPD in the cells. Because UV light-induced DNA damage is not repaired at all in nucleotide excision repair-deficient cells, proliferation of these cells after UV light irradiation must be achieved by tolerance of the damage at replication. We found that RNA interference designed to suppress polymerase zeta activity made the cells more sensitive to UV light. This increase in sensitivity was prevented by photorepair of 6-4PPs but not by photorepair of CPDs, indicating that polymerase zeta is involved in the tolerance of 6-4PPs in human cells.     

Sequence effect on incision by (A)BC excinuclease of 4NQO adducts and UV photoproducts.

Nucleotide excision repair in Escherichia coli is initiated by (A)BC excinuclease, an enzyme which incises DNA on both sides of bulky adducts and removes the damaged nucleotide as a 12-13 base long oligomer. The incision pattern of the enzyme was examined using DNA modified by 4-nitroquinoline 1-oxide (4NQO) and UV light. Similar to the cleavage pattern of UV photoproducts and other bulky adducts, the enzyme incises the 8th phosphodiester bond 5' and 5th phosphodiester bond 3' to the 4NQO-modifed base, primarily guanine. The extent of DNA damage by these agents was determined using techniques which quantitatively cleave the DNA or stop at the site of the adduct. By comparison of the intensity of gel bands created by (A)BC excinuclease and the specific cleavage at the damaged site, the efficiency of (A)BC excinuclease incision at 13 different 4NQO-induced adducts and 13 different photoproducts was determined by densitometric scanning. In general, incisions made at 4NQO-induced adducts are proportional to the extent of damage, though the efficiency of cutting throughout the sequence tested varies from 25 to 75%. Incisions made at pyrimidine dimers are less efficient than at 4NQO-adducts, ranging from 13 to 65% incision relative to modification, though most are around 50%. The two (6-4) photoproducts within the region tested are incised more efficiently than any pyrimidine dimer.     

A new mechanism for repairing oxidative damage to DNA: (A)BC excinuclease removes AP sites and thymine glycols from DNA

Escherichia coli (A)BC excinuclease is the major enzyme responsible for removing bulky adducts, such as pyrimidine dimers and 6-4 photoproducts, from DNA. Mutants deficient in this enzyme are extremely sensitive to UV and UV-mimetic agents, but not to oxidizing agents, or ionizing radiation which damages DNA in part by generating active oxygen species. DNA glycosylases and AP1 endonucleases play major roles in repairing oxidative DNA damage, and thus it has been assumed that nucleotide excision repair has no role in cellular defense against damage by ionizing radiation and oxidative damage. In this study we show that the E. coli nucleotide excision repair enzyme (A)BC excinuclease removes from DNA the two major products of oxidative damage, thymine glycol and the baseless sugar (AP site). We conclude that nucleotide excision repair is an important cellular defense mechanism against oxidizing agents.     

Escherichia coli DNA photolyase reverses cyclobutane pyrimidine dimers but not pyrimidine-pyrimidone (6-4) photoproducts

The effect of purified Escherichia coli DNA photolyase on the UV light-induced pyrimidine-pyrimidone (6-4) photoproduct and cyclobutane pyrimidine dimer was investigated in vitro using enzyme purified from cells carrying the cloned phr gene (map position, 15.7 min). Photoproducts were examined both as site-specific lesions in end-labeled DNA and as chromatographically identified products in uniformly labeled DNA. E. coli DNA photolyase removed cyclobutane dimers but had no activity on pyrimidine-pyrimidone (6-4) photoproducts. Photoreactivation can therefore be used to separate the biological effects of these two UV light-induced molecular lesions.     

Purification of a HeLa cell nuclear protein that binds selectively to DNA irradiated with ultra-violet light.

Ultraviolet (UV) light induces a variety of lesions in DNA of which the pyrimidine dimer represents the major species. Pyrimidine dimers exist as both a cyclobutane type and a 6-4' (pyrimidine-2'-one) photoproduct. We have purified a protein of M(r) approximately 125,000 from HeLa cell nuclei which binds efficiently to double-stranded DNA irradiated with UV light but not to undamaged DNA. This protein was designated UVBP1 (UV damage binding protein 1). UVBP1 did not recognise DNA damaged by cisplatin. Using oligonucleotides with a single dipyrimidine site for induction of UV photoproducts, binding of UVBP1 to a TC-containing substrate was shown to be more efficient than to substrates containing a TT, a CT or a CC pair. This binding specificity implies selective recognition of the 6-4' photoproduct. Further evidence for this was provided by the finding that hot alkali treatment of the substrate (which selectively hydrolyses 6-4' photoproducts) abrogated binding of UVBP1, whereas incubation with DNA photolyase to remove cyclobutane dimers did not. No detectable DNA helicase, ATPase or exonuclease activity was associated with the purified protein. We suggest that UVBP1 may be involved in the lesion recognition step of DNA excision repair and could contribute to the preferential repair of 6-4' photoproducts from the DNA of UV-irradiated mammalian cells.     

Substrate overlap and functional competition between human nucleotide excision repair and Escherichia coli photolyase and (a)BC excision nuclease

Human cell free extract prepared by the method of Manley et al. (1980) carries out repair synthesis on UV-irradiated DNA. Removal of pyrimidine dimers by photoreactivation with DNA photolyase reduces repair synthesis by about 50%. With excess enzyme in the reaction mixture photolyase reduced the repair signal by the same amount even in the absence of photoreactivating light, presumably by binding to pyrimidine dimers and interfering with the binding of human damage recognition protein. Similarly, the UvrB subunit of Escherichia coli (A)BC excinuclease when loaded onto UV-irradiated or psoralen-adducted DNA inhibited repair synthesis by cell-free extract by 75-80%. The opposite was true also as HeLa cell free extract specifically inhibited the photorepair of a thymine dimer by DNA photolyase and its removal by (A)BC excinuclease. Cell-free extracts from xeroderma pigmentosum (XP) complementation groups A and C were equally effective in blocking the E. coli repair proteins, while extracts from complementation groups D and E were ineffective in blocking the E. coli enzyme. These results suggest that XP-D and XP-E cells are defective in the damage recognition subunit(s) of human excision nuclease.     

Characterization of Arabidopsis photolyase enzymes and analysis of their role in protection from ultraviolet-B radiation

DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.     

Activities and incision patterns of ABC excinuclease on modified DNA containing single-base mismatches and extrahelical bases

ABC excision nuclease of Escherichia coli is a DNA repair enzyme that recognizes major helical distortions caused by bulky base adducts and incises on both sides of the adduct, thus removing the modified nucleotides in the form of a 12-13-base long oligomer. We tested the enzyme with substrates that contained unusual helical structures caused by single-base mismatches or one, three, or four extrahelical bases (loops). We find that the enzyme does not cut DNAs containing helical perturbations caused by these structures. However, when the mismatched or extrahelical bases are modified with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide, a reagent specific for unpaired G and T residues, the enzyme incises at the modified nucleotides in the regular manner. In addition, we find that when mismatches and loops are located near pyrimidine dimers and (6-4) photoproducts they do not inhibit incision at the photoproducts by the excinuclease but sometimes affect the incision pattern. Our results indicate that ABC excinuclease may be a useful enzymatic reagent to probe the structural changes caused by mismatches and deletions in DNA and provide additional information on the requirements for incision by this repair enzyme.     

Repair of the three main types of bipyrimidine DNA photoproducts in human keratinocytes exposed to UVB and UVA radiations

Induction of DNA damage by solar UV radiation is a key event in the development of skin cancers. Bipyrimidine photoproducts, including cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (64 PPs) and their Dewar valence isomers, have been identified as major UV-induced DNA lesions. In order to identify the predominant and most persistent lesions, we studied the repair of the three types of photolesions in primary cultures of human keratinocytes. Specific and quantitative data were obtained using HPLC associated with tandem mass spectrometry. As shown in other cell types, 64 PPs are removed from UVB-irradiated keratinocytes much more efficiently than CPDs. In contrast, CPDs are still present in high amounts when cells recover their proliferation capacities after cell cycle arrest and elimination of a part of the population by apoptosis. The predominance of CPDs is still maintained when keratinocytes are exposed to a combination of UVB and UVA. Under these conditions, 64 PPs are converted into their Dewar valence isomers that are as efficiently repaired as their (6-4) precursors. Exposure of cells to pure UVA radiation generates thymine cyclobutane dimers that are slightly less efficiently repaired than CPDs produced upon UVB irradiation. Altogether, our results show that CPDs are the most frequent and the less efficiently repaired bipyrimidine photoproducts irrespectively of the applied UV treatment.     

Repair of pyrimidine dimer ultraviolet light photoproducts by human cell extracts

A newly developed method allows human cell extracts to carry out repair synthesis on ultraviolet light irradiated closed circular plasmid DNA [Wood, R. D., Robins, P., & Lindahl, T. (1988) Cell 53, 97-106]. The identity of the photodamage that leads to this repair replication was investigated. Removal of stable pyrimidine hydrates from irradiated plasmid pAT153 did not significantly affect the amount of repair replication in the fluence range of 0-450 J/m2, because of the low yield of these products and their short DNA repair patch size. Photoreactivation of irradiated DNA using purified Escherichia coli DNA photolyase to remove more than 95% of the cyclobutane dimers from the DNA reduced the observed repair synthesis by 20-40%. The greater part of the repair synthesis is highly likely to be caused by (6-4) pyrimidine dimer photoproducts. This class of lesions is rapidly repaired by mammalian cells, and their removal is known to be important for cell survival after ultraviolet irradiation.     

Cyclobutane pyrimidine dimers are responsible for the vast majority of mutations induced by UVB irradiation in mammalian cells

The most prevalent DNA lesions induced by UVB are the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). It has been a long standing controversy as to which of these photoproduct is responsible for mutations in mammalian cells. Here we have introduced photoproduct-specific DNA photolyases into a mouse cell line carrying the transgenic mutation reporter genes lacI and cII. Exposure of the photolyase-expressing cell lines to photoreactivating light resulted in almost complete repair of either CPDs or (6-4)PPs within less than 3 h. The mutations produced by the remaining, nonrepaired photoproducts were scored. The mutant frequency in the cII gene after photoreactivation by CPD photolyase was reduced from 127 x 10(-5) to 34 x 10(-5) (background, 8-10 x 10(-5)). Photoreactivation with (6-4) photolyase did not lower the mutant frequency appreciably. In the lacI gene the mutant frequency after photoreactivation repair of CPDs was reduced from 148 x 10(-5) to 28 x 10(-5) (background, 6-10 x 10(-5)). Mutation spectra obtained with and without photoreactivation by CPD photolyase indicated that the remaining mutations were derived from background mutations, unrepaired CPDs, and other DNA photopoducts including perhaps a small contribution from (6-4)PPs. We conclude that CPDs are responsible for at least 80% of the UVB-induced mutations in this mammalian cell model.     

Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.     

The induction and repair of (6-4) photoproducts in Neurospora crassa.

The (6-4) photoproduct lesion found in DNA after UV irradiation is repaired by germinating Neurospora crassa conidia. Wild-type Neurospora removes 80% of the (6-4) photoproduct in approximately 20 min and maximal repair is accomplished by 30 min with approximately 89% of the original lesions removed. Mutagen-sensitive Neurospora mutants belonging to the established excision repair epistasis group, UVS-2, are not defective in the removal of cyclobutane pyrimidine dimers. Furthermore, we find these mutants capable of removing (6-4) photoproducts from their DNA at a rate similar to wild type. Comparable kinetics are also observed in key members of the other two epistasis groups.     

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.     

Effects of temperature on the photoreactivation of ultraviolet-b–induced dna damage in Palmaria palmata (Rhodophyta)

The accumulation of DNA damage (thymine dimers and 6-4 photoproducts) induced by ultraviolet-B radiation was studied in Palmaria palmata (L.) O. Kuntze under different light and temperature conditions, using specific monoclonal antibodies and subsequent chemiluminescent detection. Both types of damage were repaired much faster under ultraviolet-A radiation (UVAR) plus photosynthetically active radiation (PAR) than in darkness, which indicates photoreactivating activity. At 12° C, all thymine dimers were repaired after 2 h irradiation with UVAR plus PAR, whereas 6-4 photoproducts were almost completely repaired after 4 h. After 19 h of darkness, almost complete repair of 6-4 photoproducts was found, and 67% of the thymine dimers were repaired. In a second set of experiments, repair of DNA damage under UVAR plus PAR was compared at three different temperatures (0, 12, and 25° C). Again, thymine dimers were repaired faster than 6-4 photoproducts at all three temperatures. At 0° C, significant repair of thymine dimers was found but not of 6-4 photoproducts. Significant repair of both thymine dimers and 6-4 photoproducts occurred at 12 and 25° C. Optimal repair efficiency was found at 25° C for thymine dimers but at 12° C for 6-4 photoproducts, which suggests that the two photorepair processes have different temperature characteristics.     

Differential repair of UVB-induced cyclobutane pyrimidine dimers in cultured human skin cells and whole human skin

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are the two main classes of mutagenic DNA damages induced by UVB radiation. Numerous studies have been devoted so far to their formation and repair in human cells and skin. However, the biochemical methods used often lack the specificity that would allow the individual study of each of the four CPDs and 6-4PPs produced at TT, TC, CT and CC dinucleotides. In the present work, we applied an HPLC-mass spectrometry assay to study the formation and repair of CPDs and 6-4PPs photoproducts in primary cultures of human keratinocytes and fibroblasts as well as in whole human skin. We first observed that the yield of dimeric lesions was slightly higher in fibroblasts than in keratinocytes. In contrast, the rate of global repair was higher in the last cell type. Moreover, removal of DNA photoproducts in skin biopsies was found to be slower than in both cultured skin cells. In agreement with previous works, the repair of 6-4PPs was found to be more efficient than that of CPDs in the three types of samples, with no observed difference between the removal of the TT and TC derivatives. In contrast, a significant influence of the nature of the two modified pyrimidines was observed on the repair rate of CPDs. The decreasing order of removal efficiency was the following: C<>T>C<>C>T<>C>T<>T. These data, together with the known intrinsic mutational properties of the lesions, would support the reported UV mutation spectra. A noticeable exception concerns CC dinucleotides that are mutational hotspots with an UV-specific CC to TT tandem mutation, although related bipyrimidine photoproducts are produced in low yields and efficiently repaired.