Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

An alternative eukaryotic DNA excision repair pathway.

DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe.     

Characterization of a wide range base-damage-endonuclease activity of mammalian rpS3

Mammalian rpS3, a ribosomal protein S3 with a DNA repair endonuclease activity, nicks heavily UV-irradiated DNA and DNA containing AP sites. RpS3 calls for a novel endonucleolytic activity on AP sites generated from pyrimidine dimers by T4 pyrimidine dimer glycosylase activity. This study revealed that rpS3 cleaves the lesions including AP sites, thymine glycols, and other UV damaged lesions such as pyrimidine dimers. This enzyme does not have a glycosylase activity as predicted from its amino acid sequence. However, it has an endonuclease activity on DNA containing thymine glycol, which is exactly overlapped with UV-irradiated or AP DNAs, indicating that rpS3 cleaves phosphodiester bonds of DNAs containing altered bases with broad specificity acting as a base-damage-endonuclease. RpS3 cleaves supercoiled UV damaged DNA more efficiently than the relaxed counterpart, and the endonuclease activity of rpS3 was inhibited by MgCl2 on AP DNA but not on UV-irradiated DNA.     

Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Characterization of the alternative excision repair pathway of UV-damaged DNA in Schizosaccharomyces pombe.

Schizosaccharomyces pombe cells deficient in nucleotide excision repair (NER) are still able to remove photoproducts from cellular DNA, showing that there is a second pathway for repair of UV damage in this organism. We have characterized this repair pathway by cloning and disruption of the genomic gene encoding UV damage endonuclease (UVDE). Although uvde gene disruptant cells are only mildly UV sensitive, a double disruptant of uvde and rad13 (a S. pombe mutant defective in NER) was synergistically more sensitive than either single disruptant and was unable to remove any photoproducts from cellular DNA. Analysis of the kinetics of photoproduct removal in different mutants showed that the UVDE-mediated pathway operates much more rapidly than NER. In contrast to a previous report, our genetic analysis showed that rad12 and uvde are not the same gene. Disruption of the rad2 gene encoding a structure- specific flap endonuclease makes cells UV sensitive, but much of this sensitivity is not observed if the uvde gene is also disrupted. Further genetic and immunochemical analyses suggest that DNA incised by UVDE is processed by two separate mechanisms, one dependent and one independent of flap endonuclease.     

Isolation and characterization of nucleotide excision repair deficient mutants of the entomopathogenic fungus, Beauveria bassiana

To better understand DNA repair in the entomopathogenic fungus Beauveria bassiana, three ultraviolet (UV) light sensitive mutants were isolated and characterized to be deficient in nucleotide excision repair (NER). The UV sensitive mutants were scored by comparison to survival of the parental isolate, GK2016, after 36 J/m² UV-C irradiation. At this dose, conidial survival of GK2016 was 98% and the mutants LC75, LC194, and LC85 had survival values of 63%, 45%, and 31%, respectively. An immunological method which measured the removal of pyrimidine-(6-4)-pyrimidone photoproducts during repair confirmed the decreased ability of LC75, LC194, and LC85 to remove these UV-induced dimers by NER. The mutants were also found to be deficient in NER at swollen/ germinating conidia and blastospore life cycle stages. The germination of the moderately UV sensitive mutant, LC75, was similar to that of the parental isolate, GK2016, after UV irradiation and incubation to enhance NER. The more sensitive mutants, LC194 and LC85 were 2.1- or 2.7-fold, respectively, less likely to germinate after UV irradiation based on their ability to carry out NER. These NER deficient mutants, the first to be derived from B. bassiana, reveal the importance of NER in spore survival post-UV irradiation.     

Assessment of DNA damage and repair in Mycobacterium terrae after exposure to UV irradiation

AIM: Ultraviolet (UV) irradiation for drinking water treatment was examined for inactivation and subsequent dark and photo-repair of Mycobacterium terrae. METHODS AND RESULTS: UV sources tested were low pressure (monochromatic, 254 nm) and medium pressure (polychromatic UV output) Hg lamps. UV exposure resulted in inactivation, and was followed by dark or photo-repair experiments. Inactivation and repair were quantified utilizing a molecular-based endonuclease sensitive site (ESS) assay and conventional colony forming unit (CFU) viability assay. Mycobacterium terrae was more resistant to UV disinfection compared to many other bacteria, with approximately 2-log reduction at a UV fluence of 10 mJ cm(-2) ; similar to UV inactivation of M. tuberculosis. There was no difference in inactivation between monochromatic or polychromatic UV lamps. Mycobacterium terrae did not undergo detectable dark repair. Photo-repair resulted in recovery from inactivation by approximately 0.5-log in less than 30 min for both UV lamp systems. CONCLUSIONS: Mycobacterium terrae is able to photo-repair DNA damage within a short timeframe. The number of pyrimidine dimers induced by UV light were similar for Escherichia coli and M. terrae, however, this similarity did not hold true for viability results. SIGNIFICANCE AND IMPACT OF THE STUDY: There is no practical difference between UV sources for disinfection or prevention of DNA repair for M. terrae. The capability of M. terrae to photo-repair UV damage fairly quickly is important for wastewater treatment applications where disinfected effluent is exposed to sunlight. Finally, molecular based assay results should be evaluated with respect to differences in the nucleic acid content of the test micro-organism.     

Nucleotide excision repair in higher eukaryotes: Mechanism of primary damage recognition

Nucleotide excision repair (NER) is one of the major DNA repair pathways in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation, and bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to severe diseases, including some forms of cancer. In view of the broad substrate specificity of NER, it is of interest to study how a certain set of proteins recognizes DNA lesions in contest of a large excess of intact DNA. The review focuses on DNA damage recognition, the key and, as yet, most questionable step of NER. The main models of primary damage recognition and preincision complex assembly are considered. The model of a sequential loading of repair proteins on damaged DNA seems most reasonable in light of the available data.     

The kinetics of thymine dimer excision in ultraviolet-irradiated human cells

We have investigated the kinetics of the loss of thymine dimers from the acid-insoluble fraction of several ultraviolet (UV)-irradiated cultured human cell lines. Our results show that UV fluences between 10 and 40 J/m2 produce an average of 21-85 x 10(5) thymine dimers per cell and an eventual maximal loss per cell of 12-20 x 10(5) thymine dimers. The time for half-maximal loss of dimers ranged from 12-22 h after UV irradiation. In contrast, the time for half-maximal repair synthesis of DNA measured by autoradiography was 4.5 h. This figure agrees well with reported half-maximal repair synthesis times, which range from 0.5 to 3.6 h based on our analysis. The discrepancy in the kinetics of the loss of thymine dimers from DNA and repair synthesis is discussed in terms of possible molecular mechanisms of thymine dimer excision in vivo and in terms of possible experimental artifacts.     

Suppression of UVC-induced cell damage and enhancement of DNA repair by the fermented milk, Kefir

An aqueous extract of Kefir, fermented milk originally produced in the Caucasus mountains, suppressed morphological changes of human melanoma HMV-1 and SK-MEL cells and human normal fibroblastTIG-1 cells caused by UVC-irradiation, suggesting that UV damage can be suppressed by the Kefir extract. The addition of the Kefir extract after UVC-irradiation of HVM-1 cells resulted in a remarkable decrease in intracellular reactive oxygen species (ROS) which had been increased by UVC irradiation. The Kefir extract also stimulated unscheduled DNA synthesis and suppressed UVC-induced apoptosis of HMV-1 cells. A colony formation assay revealed that the Kefir extract rescued HMV-1 cells from cell death caused by UVC irradiation. The Kefir extract, as well as methyl methanethiosulfonate which is known to enhance the nucleotide excision repair (NER) activity, exhibited strong thymine dimer repair-enhancing activity. Epigalocatechin exhibited a weak NER activity but vitamins A, C, and E and catechin showed no NER activity. The thymine dimer repair-enhancing factors in the Kefir extract were heat-stable and assumed to be molecules with a molecular weight of less than 5000. The treatment of HMV-1 cells with the Kefir extract during or before UVC- irradiation also prevented the generation of ROS and thymine dimmer, and suppressed the apoptosis of HMV-1 cells, suggesting that application of Kefir can prevent UV damage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Pyrimidine dimer excision in surviving and nonsurviving cells of ultraviolet-irradiated cultures of Escherichia coli.

We compared dimer excision in viable and nonviable cells fractions separated from Escherichia coli B/r cultures exposed to ultraviolet (UV) irradiation. For cells grown on minimal medium with glycerol as a carbon source, both fractions from the irradiated (20 J/m2, 5% survival) culture excised 60 to 70% of the thymine dimers from prelabeled DNA within 120 min. This percentage was, within experimental error, the same as that obtained from unseparated cultures. When isolated viable and nonviable populations were given a second UV exposure (20 J/m2) both types of cells were again able to excise dimers. The UV survival curve for the isolated viable population indicates that these cells are no more sensitive to radiation than exponentially growing cells not previously exposed to UV. The extent of dimer excision after UV irradiation was also the same in viable and nonviable cells separated from cultures grown on a glucose minimal medium in which both populations excised about 85% of the dimers within 120 min. These results show that the extent of removal of pyrimidine dimer from deoxyribonucleic acid is not precisely correlated with survival of repair-competent bacterial cells after exposure to UV light.     

Nucleosome positioning,nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae

The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.     

Colcemid inhibits the rejoining of the nucleotide excision repair of UVC-induced DNA damages in Chinese hamster ovary cells

In our previous study, we found that colcemid, an inhibitor of mitotic spindle, promotes UVC-induced apoptosis in Chinese hamster ovary cells (CHO.K1). In this study, a brief treatment of colcemid on cells after but not before UV irradiation could synergistically reduce the cell viability. Although colcemid did not affect the excision of UV-induced DNA damages such as [6–4] photoproducts or cyclobutane pyrimidine dimers, colcemid accumulated the DNA breaks when it was added to cells following UV-irradiation. This colcemid effect required nucleotide excision repair (NER) since the same accumulation of DNA breaks was barely or not detected in two NER defective strains of CHO cells, UV5 or UV24. Furthermore, the colcemid effect was not due to semi-conservative DNA replication or mitosis since the colcemid-caused accumulation of DNA breaks was also seen in non-replicating cells. Moreover, colcemid inhibited rejoining of DNA breaks accumulated by hydroxyurea/cytosine arabinoside following UV irradiation. Nevertheless, colcemid did not affect the unscheduled DNA synthesis as assayed by the incorporation of bromodeoxyuridine. Taken together, our results suggest that colcemid might inhibit the step of ligation of NER pathways.     

RNA polymerase II large subunit is cleaved by caspases during DNA damage-induced apoptosis

UV radiation induces DNA lesions that are repaired by the nucleotide excision repair (NER) pathway. Cells that are NER deficient such as those derived from xeroderma pigmentosum (XP) patients are susceptible to apoptosis after 10J/m(2) UV radiation, a dose largely survivable by repair proficient cells. Herein, we report that RNA polymerase II large subunit (RNAP II-LS) undergoes caspase-mediated cleavage, yielding a 140kDa C-terminal fragment in XP lymphoblasts but not NER proficient lymphoblasts after 10J/m(2) UV irradiation. Cleavage could also be induced by cisplatin or oxaliplatin, but not transplatin, an isomer of cisplatin that does not induce DNA adducts. The cleavage of RNAP II-LS was blocked by a panel of caspase inhibitors but not by proteasomal inhibitors or inhibitors of other proteases. In vitro cleavage with caspase 8 yielded the same 140kDa RNAP II-LS fragment observed in vivo. Using site-directed mutagenesis, the RNAP II-LS cleavage site was localized to an LETD sequence ending at residue 1339, which is near its C-terminal domain.     

Use of UV endonuclease from Micrococcus luteus to monitor the progress of DNA repair in UV-irradiated human cells

A sensitive enzymatic assay has been developed to follow the progress of NDA repair in human cells exposed to UV radiation. The assay employs an endonuclease selectively active at sites containing pyrimidine dimers in UV-damaged DNA. Primary fibroblasts are exposed to 254 nm radiation and incubated for specified times, their radioactivity labelled DNA is isolated and treated with a UV endonuclease extensively purified from Micrococcus luteus. Endonuclease-susceptible site remaining in the DNA are subsequently observed as single-strand scissions by sedimentation in alkaline sucrose gradients. In comparison to the situation with excision-proficient normal cells, those derived from patients suffering from either the classical or the De Sanctis-Cacchione clinical form of Xeroderma pigmentosum (XP) exhibit a marked diminution in the rate of disappearance of nuclease-susceptible lesions with time of post-UV incubation.     

Two budding yeast RAD4 homologs in fission yeast play different roles in the repair of UV-induced DNA damage

We have identified two fission yeast homologs of budding yeast Rad4 and human xeroderma pigmentosum complementation group C (XP-C) correcting protein, designated Rhp4A and Rhp4B. Here we show that the rhp4 genes encode NER factors that are required for UV-induced DNA damage repair in fission yeast. The rhp4A-deficient cells but not the rhp4B-deficient cells are sensitive to UV irradiation. However, the disruption of both rhp4A and rhp4B resulted in UV sensitivity that was greater than that of the rhp4A-deficient cells, revealing that Rhp4B plays a role in DNA repair on its own. Fission yeast has two pathways to repair photolesions on DNA, namely, nucleotide excision repair (NER) and UV-damaged DNA endonuclease-dependent excision repair (UVER). Studies with the NER-deficient rad13 and the UVER-deficient (Delta)uvde mutants showed the two rhp4 genes are involved in NER and not UVER. Assessment of the ability of the various mutants to remove cyclobutane pyrimidine dimers (CPDs) from the rbp2 gene locus indicated that Rhp4A is involved in the preferential repair of lesions on the transcribed DNA strand and plays the major role in fission yeast NER. Rhp4B in contrast acts as an accessory protein in non-transcribed strand (NTS) repair.     

Alternative Excision Repair Pathways

Alternative excision repair (AER) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of DNA damage, and followed by excision of the damaged DNA, repair synthesis, and ligation. The ultraviolet (UV) damage endonuclease in fungi and bacteria introduces a nick immediately 5′ to various types of UV damage and initiates its excision repair that is independent of nucleotide excision repair (NER). Endo IV-type apurinic/apyrimidinic (AP) endonucleases from Escherichia coli and yeast and human Exo III-type AP endonuclease APEX1 introduce a nick directly and immediately 5′ to various types of oxidative base damage besides the AP site, initiating excision repair. Another endonuclease, endonuclease V from bacteria to humans, binds deaminated bases and cleaves the phosphodiester bond located 1 nucleotide 3′ of the base, leading to excision repair. A single-strand break in DNA is one of the most frequent types of DNA damage within cells and is repaired efficiently. AER makes use of such repair capability of single-strand breaks, removes DNA damage, and has an important role in complementing BER and NER.NER and base excision repair (BER) are the major excision repair pathways present in almost all organisms. In NER, dual incisions are introduced, the damaged DNA between the incised sites is then removed, and DNA synthesis fills the single-stranded gap, followed by ligation. In BER, an AP site, formed by depurination or created by a base damage-specific DNA glycosylase, is recognized by an AP endonuclease that introduces a nick immediately 5′ to the AP site, followed by repair synthesis, removal of the AP site, and final ligation. Besides these two fundamental excision repair systems, investigators have found another category of excision repair—AER—an example of which is the excision repair of UV damage, initiated by an endonuclease called UV damage endonuclease (UVDE). UVDE introduces a single nick immediately 5′ to various types of UV lesions as well as other types of base damage, and this nick leads to the removal of the lesions by an AER process designated as UVDE-mediated excision repair (UVER or UVDR). Genetic analysis in Schizosaccharomyces pombe indicates that UVER provides cells with an extremely rapid removal of UV lesions, which is important for cells exposed to UV in their growing phase.Endo IV–type AP endonucleases from Escherichia coli and budding yeast and the Exo III–type human AP endonuclease APEX1 are able to introduce a nick at various types of oxidative base damage and initiate a form of excision repair that has been designated as nucleotide incision repair (NIR). Endonuclease V (ENDOV) from bacteria to humans recognizes deaminated bases, introduces a nick 1 nucleotide 3′ of the base, and leads to excision repair initiated by the nick. These endonucleases introduce a single nick near the DNA-damage site, leaving 3′-OH termini, and initiate repair of both the DNA damage and the nick. The mechanisms of AER may be similar to those of single-strand break (SSB) repair or BER except for the initial nicking process. However, how DNA damage is recognized determines the repair process within the cell. This article discusses the mechanisms and functional roles of AER. We begin with AER of UV damage, because genetic analysis has shown functional differences between this AER and NER in S. pombe.     

Repair of DNA Damage Induced by Solar UV

Solar UV radiation induces significant levels of DNA damage in living things. This damage, if left unrepaired, is lethal in humans. Recent work has demonstrated that plants possess several repair pathways for UV-induced DNA damage, including pathways for the photoreactivation of both 6-4 products and cyclobutane pyrimidine dimers (CPDs), the two lesions most frequently induced by UV. Plants also possess the more general nucleotide excision repair (NER) pathway as well as bypass polymerases that enable the plant to replicate its DNA in the absence of DNA repair.This revised version was published online in October 2005 with corrections to the Cover Date.