AtRAD1, a plant homologue of human and yeast nucleotide excision repair endonucleases, is involved in dark repair of UV damages and recombination

Plants are unique in the obligatory nature of their exposure to sunlight and consequently to ultraviolet (UV) irradiation. However, our understanding of plant DNA repair processes lags far behind the current knowledge of repair mechanisms in microbes, yeast and mammals, especially concerning the universally conserved and versatile dark repair pathway called nucleotide excision repair (NER). Here we report the isolation and functional characterization of Arabidopsis thaliana AtRAD1, which encodes the plant homologue of Saccharomyces cerevisiae RAD1, Schizosaccharomyces pombe RAD16 and human XPF, endonucleolytic enzymes involved in DNA repair and recombination processes. Our results indicate that AtRAD1 is involved in the excision of UV-induced damages, and allow us to assign, for the first time in plants, the dark repair of such DNA lesions to NER. The low efficiency of this repair mechanism, coupled to the fact that AtRAD1 is ubiquitously expressed including tissues that are not accessible to UV light, suggests that plant NER has other roles. Possible 'UV-independent' functions of NER are discussed with respect to features that are particular to plants.     

Arabidopsis UVH3 gene is a homolog of the Saccharomyces cerevisiae RAD2 and human XPG DNA repair genes

To identify mechanisms of DNA repair in Arabidopsis thaliana, we have analyzed a mutant (uvh3) which exhibits increased sensitivity to ultraviolet (UV) light, H2O2 and ionizing radiation and displays a premature senescence phenotype. The uvh3 locus was mapped within chromosome III to the GL1 locus. A cosmid contig of the GL1 region was constructed, and individual cosmids were used to transform uvh3 mutant plants. Cosmid N9 was found to confer UV-resistance, H2O2-resistance and a normal senescence phenotype following transformation, indicating that the UVH3 gene is located on this cosmid and that all three phenotypes are due to the same mutation. Analysis of cosmid N9 sequences identified a gene showing strong similarity to two homologous repair genes, RAD2 (Saccharomyces cerevisiae) and XPG (human), which encode an endonuclease required for nucleotide excision repair of UV-damage. The uvh3 mutant was shown to carry a nonsense mutation in the coding region of the AtRAD2/XPG gene, thus revealing that the UVH3 gene encodes the AtRAD2/XPG gene product. In humans, the homologous XPG protein is also involved in removal of oxygen-damaged nucleotides by base excision repair. We discuss the possibility that the increased sensitivity of the uvh3 mutant to H2O2 and the premature senescence phenotype might result from failure to repair oxygen damage in plant tissues. Finally, we show that the AtRAD2/XPG gene is expressed at moderate levels in all plant tissues.     

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.     

Mutations at the mei-41, mus(1)101, mus(1)103, mus(2)205 and mus(3)310 loci of Drosophila exhibit differential UDS responses with different DNA-damaging agents

5 mutagen-sensitive mutants of Drosophila melanogaster, reported to perform normal or only slightly reduced excision repair of UV damage, were examined by an unscheduled DNA synthesis (UDS) assay. This assay measures the ability of cultured primary cells, derived from each mutant, to perform the resynthesis step in the excision repair pathway, following damage to cellular DNA by direct-acting alkylating agents, UV or X-irradiation. 2 mutants, classified as completely or partially proficient for both excision and postreplication repair of UV damage, mus(1)103 and mus(2)205, were found to give positive UDS responses only for UV damage. These mutants exhibit no measurable UDS activity following DNA damage by several different alkylating agents and X-rays. 3 mutants, classified as having no defect in excision repair, but measurable defects in postreplication repair of UV damage, mei-41, mus(1)101, and mus(3)310 exhibit 3 different response patterns when tested with the battery of agents in the UDS assay. The mutant mei-41 exhibits a highly positive UDS response following damage by all agents, consistent with its prior classification as excision-repair-proficient, but postreplication-repair-deficient for UV damage. The mutant mus(1)101, however, exhibits a strong positive UDS response following only UV damage and appears to be blocked in the excision repair of damage produced by both alkylating agents and X-irradiation. Finally, mus(3)310 exhibits no UDS response to alkylation, X-ray or UV damage. This is not consistent with its previous classification. Results obtained with the quantitative in vitro UDS assay are entirely consistent with the results from two separate in vivo measures of excision repair deficiency following DNA damage, larval hypersensitivity to killing and hypermutability in the sex-linked recessive lethal test.     

Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines.

DNA polymerase beta is required in mammalian cells for the predominant pathway of base excision repair involving single nucleotide gap filling DNA synthesis. Here we examine the relationship between oxidative stress, cellular levels of DNA polymerase beta and base excision repair capacity in vitro , using mouse monocytes and either wild-type mouse fibroblasts or those deleted of the DNA polymerase beta gene. Treatment with an oxidative stress-inducing agent such as hydrogen peroxide, 3-morpholinosydnonimine, xanthine/xanthine oxidase or lipopolysaccharide was found to increase the level of DNA polymerase beta in both monocytes and fibroblasts. Base excision repair capacity in vitro , as measured in crude cell extracts, was also increased by lipopolysaccharide treatment in both cell types. In monocytes lipopolysaccharide-mediated up-regulation of the base excision repair system correlated with increased resistance to the monofunctional DNA alkylating agent methyl methanesulfonate. By making use of a quantitative PCR assay to detect lesions in genomic DNA we show that lipopolysaccharide treatment of fibroblast cells reduces the incidence of spontaneous DNA lesions. This effect may be due to the enhanced DNA polymerase beta-dependent base excision repair capacity of the cells, because a similar decrease in DNA lesions was not observed in cells deficient in base excision repair by virtue of DNA polymerase beta gene deletion. Similarly, fibroblasts treated with lipopolysaccharide were more resistant to methyl methanesulfonate than untreated cells. This effect was not observed in cells deleted of the DNA polymerase beta gene. These results suggest that the DNA polymerase beta-dependent base excision repair pathway can be up-regulated by oxidative stress-inducing agents in mouse cell lines.     

Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts.

Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.     

Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1

To analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium-mediated transformation of the uvh1-1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1-1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1-1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3' splice site and introducing an in-frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues.     

Efficient in vitro repair of 7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple pathways.

To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.     

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.     

Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance

DNA double strand breaks (DSBs) are one of the most cytotoxic forms of DNA damage and must be repaired by recombination, predominantly via non-homologous joining of DNA ends (NHEJ) in higher eukaryotes. However, analysis of DSB repair kinetics of plant NHEJ mutants atlig4-4 and atku80 with the neutral comet assay shows that alternative DSB repair pathways are active. Surprisingly, these kinetic measurements show that DSB repair was faster in the NHEJ mutant lines than in wild-type Arabidopsis.Here we provide the first characterization of this KU-independent, rapid DSB repair pathway operating in Arabidopsis. The alternate pathway that rapidly removes the majority of DSBs present in nuclear DNA depends upon structural maintenance of chromosomes (SMC) complex proteins, namely MIM/AtRAD18 and AtRAD21.1. An absolute requirement for SMC proteins and kleisin for rapid repair of DSBs in Arabidopsis opens new insight into the mechanism of DSB removal in plants.     

Functional conservation of the yeast and Arabidopsis RAD54-like genes

The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance severalfold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54Delta mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.     

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.     

Dominant negative rat DNA polymerase beta mutants interfere with base excision repair in Saccharomyces cerevisiae.

DNA polymerase beta is one of the smallest known eukaryotic DNA polymerases. This polymerase has been very well characterized in vitro, but its functional role in vivo has yet to be determined. Using a novel competition assay in Escherichia coli, we isolated two DNA polymerase beta dominant negative mutants. When we overexpressed the dominant negative mutant proteins in Saccharomyces cerevisiae, the cells became sensitive to methyl methanesulfonate. Interestingly, overexpression of the same polymerase beta mutant proteins did not confer sensitivity to UV damage, strongly suggesting that the mutant proteins interfere with the process of base excision repair but not nucleotide excision repair in S. cerevisiae. Our data implicate a role for polymerase IV, the S. cerevisiae polymerase beta homolog, in base excision repair in S. cerevisiae.     

CENTRIN2 Interacts with the Arabidopsis Homolog of the Human XPC Protein (AtRAD4) and Contributes to Efficient Synthesis-dependent Repair of Bulky DNA Lesions

Arabidopsis thaliana CENTRIN2 (AtCEN2) has been shown to modulate Nucleotide Excision Repair (NER) and Homologous Recombination (HR). The present study provides evidence that AtCEN2 interacts with the Arabidopsis homolog of human XPC, AtRAD4 and that the distal EF-hand Ca²⁺ binding domain is essential for this interaction. In addition, the synthesis-dependent repair efficiency of bulky DNA lesions was enhanced in cell extracts prepared from Arabidopsis plants overexpressing the full length AtCEN2 but not in those overexpressing a truncated AtCEN2 form, suggesting a role for the distal EF-hand Ca²⁺ binding domain in the early step of the NER process. Upon UV-C treatment the AtCEN2 protein was shown to be increased in concentration and to be localised in the nucleus rapidly. Taken together these data suggest that AtCEN2 is a part of the AtRAD4 recognition complex and that this interaction is required for efficient NER. In addition, NER and HR appear to be differentially modulated upon exposure of plants to DNA damaging agents. This suggests in plants, that processing of bulky DNA lesions highly depends on the excision repair efficiency, especially the recognition step, thus influencing the recombinational repair pathway.     

DNA polymerase lambda protects mouse fibroblasts against oxidative DNA damage and is recruited to sites of DNA damage/repair

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.     

Human nucleotide excision repair in vitro: repair of pyrimidine dimers, psoralen and cisplatin adducts by HeLa cell-free extract.

We searched for nucleotide excision repair in human cell-free extracts using two assays: damage-specific incision of DNA (the nicking assay) and damage-stimulated DNA synthesis (the repair synthesis assay). HeLa cell-free extract prepared by the method of Manley et al. (1980) has a weak nicking activity on UV irradiated DNA and the nicking is only slightly reduced when pyrimidine dimers are eliminated from the substrate by DNA photolyase. In contrast to the nicking assay, the extract gives a strong signal with UV irradiated substrate in the repair synthesis assay. The repair synthesis activity is ATP dependent and is reduced by about 50% by prior treatment of the substrate with DNA photolyase indicating that this fraction of repair synthesis is due to removal of pyrimidine dimers by nucleotide excision. Psoralen and cisplatin adducts which are known to be removed by nucleotide excision repair also elicited repair synthesis activity 5-10 fold above the background synthesis. When M13RF DNA containing a uniquely placed psoralen adduct was used in the reaction, complete repair was achieved in a fraction of molecules as evidenced by the restoration of psoralen inactivated KpnI restriction site. This activity is absent in xeroderma pigmentosum group A cells. We conclude that our cell-free extract contains the human nucleotide excision repair enzyme activity.     

The DNA translocase RAD5A acts independently of the other main DNA repair pathways,and requires both its ATPase and RING domain for activity in Arabidopsis thaliana

Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error‐free branch of post‐replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication‐associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (AtRAD1), single‐strand break repair (AtPARP1), as well as microhomology‐mediated double‐strand break repair (AtTEB). Moreover, RAD5A can partially complement for a deficient AtATM‐mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects.     

A repair competition assay to assess recognition by human nucleotide excision repair.

We developed a competition assay to compare, in a quantitative manner, the ability of human nucleotide excision repair (NER) to recognise structurally different forms of DNA damage. This assay uses a NER substrate consisting of M13 double-stranded DNA with a single and uniquely located acetylaminofluorene (AAF) adduct, and measures the efficiency by which multiply damaged plasmid DNA competes for excision repair of the site-directed modification. To validate this assay, we tested competitor DNA containing defined numbers of either AAF adducts or UV radiation products. In both cases, repair of the site-directed NER substrate was inhibited in a damage-specific and dose-dependent manner. We then exploited this competition assay to determine the susceptibility of bulky adozelesin-DNA adducts to human NER.