Mitochondrial DNA repair by photolyase.

Photolyase genes of Saccharomyces cerevisiae and Escherichia coli were expressed in S. cerevisiae and photoreactivation in nuclei and mitochondria of the host cells was analyzed by determination of survival and petit rates. Yeast photolyase was able to repair mitochondrial DNA effectively, whereas E. coli photolyase could reduce only a small fraction of the petit rate produced by UV irradiation. Analysis using fusion between yeast photolyase and E. coli lacZ genes as well as a chimeric gene between yeast and E. coli photolyase genes suggests the importance of the protruding amino terminal region of the yeast photolyase for its transport into mitochondria. A significant similarity between the protruding amino termini of yeast photolyase and yeast uracil-DNA-glycosylase suggests a common functional importance of the terminal sequences for both DNA repair enzymes.     

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.     

Repair of UV lesions in nucleosomes--intrinsic properties and remodeling

Nucleotide excision repair and reversal of pyrimidine dimers by photolyase (photoreactivation) are two major pathways to remove UV-lesions from DNA. Here, it is discussed how lesions are recognized and removed when the DNA is condensed into nucleosomes. During the recent years it was shown that nucleosomes inhibit photolyase and excision repair in vitro and slow down repair in vivo. The correlation of DNA-repair rates with nucleosome positions in yeast suggests that intrinsic properties of nucleosomes such as mobility and transient unwrapping of nucleosomal DNA facilitate damage recognition. Moreover, it was shown that nucleosome remodeling activities can act on UV-damaged DNA in vitro and facilitate repair suggesting that random remodeling of chromatin might contribute to damage recognition in vivo. Recent work on nucleosome structure and mobility is included to evaluate how nucleosomes accommodate DNA lesions and how nucleosome mobility and remodeling can take place on damaged DNA.     

Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli.

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.     

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.     

Evidence that xeroderma pigmentosum cells from complementation group E are deficient in a homolog of yeast photolyase.

Xeroderma pigmentosum (XP) patients are deficient in the excision repair of damaged DNA. Recognition of the DNA lesion appears to involve a nuclear factor that is defective in complementation group E (XPE binding factor). We have now identified a factor in the yeast Saccharomyces cerevisiae that shares many properties with XPE binding factor, including cellular location, abundance, magnesium dependence, and relative affinities for multiple forms of damaged DNA. Yeast binding activity is dependent on photolyase, which catalyzes the photoreactivation of pyrimidine dimers. These results suggest that yeast photolyase may also function as an auxiliary protein in excision repair. Furthermore, XPE binding factor appears to be the human homolog of yeast photolyase.     

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.     

DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair

DNA damage formation and repair are tightly linked to protein–DNA interactions in chromatin. We have used minichromosomes in yeast as chromatin substrates in vivo to investigate how nucleotide excision repair (NER) and repair by DNA-photolyase (photoreactivation) remove pyrimidine dimers from an origin of replication (ARS1). The ARS1 region is nuclease sensitive and flanked by nucleosomes on both sides. Photoreactivation was generally faster than NER at all sites. Site-specific heterogeneity of repair was observed for both pathways. This heterogeneity was different for NER and photoreactivation and it was altered in a minichromosome where ARS1 was transcribed. The results indicate distinct interactions of the repair systems with protein complexes bound in the ARS region (ORC, Abf1) and a predominant role of photolyase in CPD repair of an origin of replication.     

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.     

DNA photolyase homologs are the major UV resistance factors in the cyanobacterium Synechocystis sp. PCC 6803

In this study, the unicellular photosynthetic cyanobacterium Synechocystis sp. PCC 6803 was used as a model phototroph to study the contribution of enzymatic photoreactivation to the overall protection against UV irradiation. We have isolated genes encoding two DNA photolyase homologs, phrA and phrB, from Synechocystis 6803. phrA encodes an 8-hydroxy-5-deazariboflavin (HDF) type, Class I DNA photolyase. By complementing a photolyase-deficient mutant strain of Escherichia coli, we demonstrated that PhrA is a DNA photolyase. Analysis of a phrA knockout mutant strain suggested that this gene is responsible for the majority of the observed UV resistance in Synechocystis 6803. Similar studies on phrB demonstrated that it also contributes to photoreactivation, but to a much lesser degree. Based on these findings, we conclude that enzymatic photoreactivation is the primary process used for repairing UV-induced damage in Synechocystis 6803.     

An enzyme similar to animal type II photolyases mediates photoreactivation in Arabidopsis.

The important issue of photoreactivation DNA repair in plants has become even more interesting in recent years because a family of genes that are highly homologous to photoreactivating DNA repair enzymes but that function as blue light photoreceptors has been isolated. Here, we report the isolation of a novel photolyase-like sequence from Arabidopsis designated PHR1 (for photoreactivating enzyme). It shares little sequence similarity with either type I photolyases or the cryptochrome family of blue light photoreceptors. Instead, the PHR1 gene encodes an amino acid sequence with significant homology to the recently characterized type II photolyases identified in a number of prokaryotic and animal systems. PHR1 is a single-copy gene and is not expressed in dark-grown etiolated seedlings: the message is light inducible, which is similar to the expression profile for photoreactivation activity in plants. The PHR1 protein complements a photolyase-deficient mutant of Escherichia coli and thus confers photoreactivation activity. In addition, an Arabidopsis mutant that is entirely lacking in photolyase activity has been found to contain a lesion within this Arabidopsis type II photolyase sequence. We conclude that PHR1 represents a genuine plant photolyase gene and that the plant genes with homology to type I photolyases (the cryptochrome family of blue light photoreceptors) do not contribute to photoreactivation repair, at least in the case of Arabidopsis.     

Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair

Nucleotide excision repair (NER) and DNA repair by photolyase in the presence of light (photoreactivation) are the major pathways to remove UV-induced DNA lesions from the genome, thereby preventing mutagenesis and cell death. Photoreactivation was found in many prokaryotic and eukaryotic organisms, but not in mammals, while NER seems to be universally distributed. Since packaging of eukaryotic DNA in nucleosomes and higher order chromatin structures affects DNA structure and accessibility, damage formation and repair are coupled intimately to structural and dynamic properties of chromatin. Here, I review recent progress in the study of repair of chromatin and transcribed genes. Photoreactivation and NER are discussed as examples of how an individual enzyme and a complex repair pathway, respectively, access DNA lesions in chromatin and how these two repair processes fulfil complementary roles in removal of UV lesions. These repair pathways provide insight into the structural and dynamic properties of chromatin and suggest how other DNA repair processes could work in chromatin.     

Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers.

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores. Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S. cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S. cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E. coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate. We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA. Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer. These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair. The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer.     

DNA photolyases: physical properties, action mechanism, and roles in dark repair

DNA photolyases catalyze the light-dependent repair of cis,syn-cyclobutane dipyrimidines (pyrimidine dimers). Although the phenomenon of enzymatic photoreactivation was first described 40 years ago and photolyases were the first enzymes shown unequivocally to effect DNA repair, it has only been in the last 8 years that sufficient quantities of the enzymes have been purified to permit detailed studies of their physical properties, identification of their intrinsic chromophores, and elucidation of the mechanisms of dimer recognition and photolysis. In addition several of the genes encoding these enzymes have now been cloned and sequenced. These studies have revealed remarkable functional and structural conservation among these evolutionarily ancient enzymes and have identified a new role for photolyases in dark-repair processes which has implications for the mechanism of nucleotide excision repair in both prokaryotes and eukaryotes.     

DNA repair in higher plants; photoreactivation is the major DNA repair pathway in non-proliferating cells while excision repair (nucleotide excision repair and base excision repair) is active in proliferating cells

We investigated expression patterns of DNA repair genes such as the CPD photolyase, UV-DDB1, CSB, PCNA, RPA32 and FEN-1 genes by northern hybridization analysis and in situ hybridization using a higher plant, rice (Oryza sativa L. cv. Nipponbare). We found that all the genes tested were expressed in tissues rich in proliferating cells, but only CPD photolyase was expressed in non-proliferating tissue such as the mature leaves and elongation zone of root. The removal of DNA damage, cyclobutane pyrimidine dimers and (6–4) photoproducts, in both mature leaves and the root apical meristem (RAM) was observed after UV irradiation under light. In the dark, DNA damage in mature leaves was not repaired efficiently, but that in the RAM was removed rapidly. Using a rice 22K custom oligo DNA microarray, we compared global gene expression patterns in the shoot apical meristem (SAM) and mature leaves. Most of the excision repair genes were more strongly expressed in SAM. These results suggested that photoreactivation is the major DNA repair pathway for the major UV-induced damage in non-proliferating cells, while both photoreactivation and excision repair are active in proliferating cells.     

Cyclobutane pyrimidine dimer (CPD) photolyase repairs ultraviolet-B-induced CPDs in rice chloroplast and mitochondrial DNA

Plants use sunlight as energy for photosynthesis; however, plant DNA is exposed to the harmful effects of ultraviolet‐B (UV‐B) radiation (280–320 nm) in the process. UV‐B radiation damages nuclear, chloroplast and mitochondrial DNA by the formation of cyclobutane pyrimidine dimers (CPDs), which are the primary UV‐B‐induced DNA lesions, and are a principal cause of UV‐B‐induced growth inhibition in plants. Repair of CPDs is therefore essential for plant survival while exposed to UV‐B‐containing sunlight. Nuclear repair of the UV‐B‐induced CPDs involves the photoreversal of CPDs, photoreactivation, which is mediated by CPD photolyase that monomerizes the CPDs in DNA by using the energy of near‐UV and visible light (300–500 nm). To date, the CPD repair processes in plant chloroplasts and mitochondria remain poorly understood. Here, we report the photoreactivation of CPDs in chloroplast and mitochondrial DNA in rice. Biochemical and subcellular localization analyses using rice strains with different levels of CPD photolyase activity and transgenic rice strains showed that full‐length CPD photolyase is encoded by a single gene, not a splice variant, and is expressed and targeted not only to nuclei but also to chloroplasts and mitochondria. The results indicate that rice may have evolved a CPD photolyase that functions in chloroplasts, mitochondria and nuclei, and that contains DNA to protect cells from the harmful effects of UV‐B radiation.