Repair of (6‐4) Lesions in DNA by (6‐4) Photolyase: 20 Years of Quest for the Photoreaction Mechanism

Exposure of DNA to ultraviolet (UV) light from the Sun or from other sources causes the formation of harmful and carcinogenic crosslinks between adjacent pyrimidine nucleobases, namely cyclobutane pyrimidine dimers and pyrimidine(6–4)pyrimidone photoproducts. Nature has developed unique flavoenzymes, called DNA photolyases, that utilize blue light, that is photons of lower energy than those of the damaging light, to repair these lesions. In this review, we focus on the chemically challenging repair of the (6–4) photoproducts by (6–4) photolyase and describe the major events along the quest for the reaction mechanisms, over the 20 years since the discovery of (6‐4) photolyase.     

UNIQUE DNA REPAIR PROPERTY OF AN ULTRAVIOLET-SENSITIVE (radC MUTANT OF Dictyostelium discoideum

Abstract— Dictyostelium discoideum is an organism that shows higher UV resistance than other organisms, such as Escherichia coli and human cultured cells. We examined the removal of cyclobutane pyrimidine dimers (CPD) and 6–4 photoproducts from DNA in the radC mutant and the wild-type strain using an enzyme-linked immunosorbent assay with monoclonal antibodies. Wild-type cells excised more than 90% of both CPD and 6–4 photoproducts within 4 h. Dictyostelium discoideum appeared to have a special repair system, because 6–4 photoproducts were repaired faster than CPD in E. coli and human cultured cells. In radC mutant cells, although only 50% of CPD were excised from DNA within 8 h, effective removal of 6–4 photoproducts (80% in 8 h) was observed. Excision repair-deficient mutants generally cannot remove both CPD and 6–4 photoproducts. Though the radC mutant shows deficient excision repair, it can remove 6–4 photoproducts to a moderate degree. These results suggest that D. discoideum has two kinds of repair systems, one mainly for CPD and the other for 6–4 photoproducts, and that the radC mutant has a defect mainly in the repair enzyme for CPD.     

DNA光复活作用机理的研究进展*

"环丁烷型嘧啶二聚体(Pyr< > Pyr) 是太阳光中紫外线造成DNA 损伤的主要光化学产物。DNA 光复活酶(或称光解酶) 能够利用可见光裂解二聚体的环丁烷环而修复DNA。本文对DNA 光复活过程中的光解酶对Pyr< > Pyr 的识别和光催化Pyr< > Pyr 裂解反应进行了综述, 介绍了DNA 光解酶的结构、DNA 的主要UV 光化学产物。较详尽地评述了国际上在光解酶催化二聚体裂解的途径以及模型研究方面的最新进展, 并预测了该领域的发展前景。     

Crystal Structure of the T(6‐4)C Lesion in Complex with a (6‐4) DNA Photolyase and Repair of UV‐Induced (6‐4) and Dewar Photolesions

UV‐light irradiation induces the formation of highly mutagenic lesions in DNA, such as cis‐syn cyclobutane pyrimidine dimers (CPD photoproducts), pyrimidine(6‐4)pyrimidone photoproducts ((6‐4) photoproducts) and their Dewar valence isomers ((Dew) photoproducts). Here we describe the synthesis of defined DNA strands containing these lesions by direct irradiation. We show that all lesions are efficiently repaired except for the T(Dew)T lesion, which cannot be cleaved by the repair enzyme under our conditions. A crystal structure of a T(6‐4)C lesion containing DNA duplex in complex with the (6‐4) photolyase from Drosophila melanogaster provides insight into the molecular recognition event of a cytosine derived photolesion for the first time. In light of the previously postulated repair mechanism, which involves rearrangement of the (6‐4) lesions into strained four‐membered ring repair intermediates, it is surprising that the not rearranged T(6‐4)C lesion is observed in the active site. The structure, therefore, provides additional support for the newly postulated repair mechanism that avoids this rearrangement step and argues for a direct electron injection into the lesion as the first step of the repair reaction performed by (6‐4) DNA photolyases.     

Photoreactivating Enzyme for (6–4) Photoproducts in Cultured Goldfish Cells

We previously reported that when cultured goldfish cells are illuminated with fluorescent light, photorepair ability for both cyclobutane pyrimidine dimers and (6–4) photoproducts increased. In the present study, it was found that the duration of the induced photorepair ability for cyclobutane pyrimidine dimers was longer than that for (6–4) photoproducts, suggesting the presence of different photolyases for repair of these two major forms of DNA damage. A gel shift assay was then performed to show the presence of protein(s) binding to (6–4) photoproducts and its dissociation from (6–4) photoproducts under fluorescent light illumination. In addition, at 8 h after fluorescent light illumination of the cell, the binding of pro-tein(s) to (6–4) photoproducts increased. The restriction enzymes that have recognition sites containing TT or TC sequences failed to digest the UV-irradiated DNA pho-toreactivated by using Escherichia coli photolyase for cyclobutane pyrimidine dimers, indicating that restriction enzymes could not function because (6–4) photoproducts remained in recognition sites. But, when UV-irradiated DNA depleted of cyclobutane pyrimidine dimers was incubated with extract of cultured goldfish cells under fluorescent light illumination, it was digested with those restriction enzymes. These results suggested the presence of (6–4) photolyase in cultured goldfish cells as in Dro-sophila, Xenopus and Crotalus.     

Photolyase: Dynamics and Mechanisms of Repair of Sun‐Induced DNA Damage

Photolyase, a photomachine discovered half a century ago for repair of sun‐induced DNA damage of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6‐4) pyrimidone photoproducts (6‐4PPs), has been characterized extensively in biochemistry (function), structure and dynamics since 1980s. The molecular mechanism and repair photocycle have been revealed at the most fundamental level. Using femtosecond spectroscopy, we have mapped out the entire dynamical evolution and determined all actual timescales of the catalytic processes. Here, we review our recent efforts in studies of the dynamics of DNA repair by photolyases. The repair of CPDs in three life kingdoms includes seven electron transfer (ET) reactions among 10 elementary steps through initial bifurcating ET pathways, a direct tunneling route and a two‐step hopping path both through an intervening adenine from the cofactor to CPD, with a conserved folded structure at the active site. The repair of 6‐4PPs is challenging and requires similar ET reactions and a new cyclic proton transfer with a conserved histidine residue at the active site of (6‐4) photolyases. Finally, we also summarize our efforts on multiple intraprotein ET of photolyases in different redox states and such mechanistic studies are critical to the functional mechanism of homologous cryptochromes of blue‐light photoreceptors.     

Identification and Purification of the CPD Photolyase in Vibrio parahaemolyticus RIMD2210633

Photoreactivation is an error‐free mechanism of DNA repair, utilized by prokaryotes and most eukaryotes and is catalyzed by specific enzymes called DNA photolyases. Photoreactivation has been reported in Vibrio parahaemolyticus WP28; however, information on photolyases in V. parahaemolyticus (V.p) strains has not been reported. This study examined the photoreactivation in V.p RIMD2210633. The photolyase responsible for repairing cyclobutane pyrimidine dimer (CPD) in DNA was identified, and the corresponding gene was determined as VPA1471. The protein was overexpressed in Escherichia coli and was purified for functional assessment in vitro. The mRNA level and protein expression level of this gene increased after ultraviolet A (UVA) illumination following ultraviolet C (UVC) irradiation. In vitro experiments confirmed that the protein encoded by VPA1471 could reduce the quantity of CPD in DNA. We designated the corresponding gene and protein of VPA1471 phr and Phr, respectively, although the function of two other photolyase/cryptochrome family members, VPA0203 and VPA0204, remains unclear. UV (ultraviolet) irradiation experiments suggest that these two genes possess some photorepairing ability. Therefore, we hypothesize that VPA0203 and VPA0204 encode (6‐4) photolyase in V. parahaemolyticus RIMD2210633.     

PREFERENTIAL INHIBITION OF NUCLEOSOME ASSEMBLY BY ULTRAVIOLET-INDUCED (6-4)PHOTOPRODUCTS

We reconstituted nucleosomes in vitro using two kinds of damaged pBR322 plasmid DNA carrying cyclobutane pyrimidine dimers (CPD) or (6-4)photoproducts. The results indicate that nucleosome assembly is inhibited preferentially by (6-4)photoproducts compared with CPD, suggesting that the regions carrying (6-4)photoproducts retain their nucleosome-free form, i.e. linker-like conformation until completion of the repair processes.     

Nucleotide excision repair proteins rapidly accumulate but fail to persist in human XP-E (DDB2 mutant) cells

The xeroderma pigmentosum (XP-E) DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from four newly reported XP-E patients with numerous skin cancers and DDB2 mutations had slow repair of 6-4 photoproducts (6-4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA and XPF) colocalized to CPD and 6-4PP positive regions immediately (<0.1 h) after localized UV irradiation in cells from the XP-E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5 h these repair proteins were no longer detectable at the sites of DNA damage in XP-E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6-4PP but is essential for normal persistence of these proteins for CPD photoproduct removal.     

DNA Ligases I and III Support Nucleotide Excision Repair in DT40 Cells with Similar Efficiency

In eukaryotic cells helix‐distorting DNA lesions like cyclobutane pyrimidine dimers (CPDs) and 6–4 pyrimidine‐pyrimidone photoproducts (6–4 PPs) are efficiently removed by nucleotide excision repair (NER). NER is a multistep process where in the end, subsequent to replication over the gap, the remaining nick is sealed by a DNA ligase. Lig1 has been implicated as the major DNA ligase in NER. Recently, Lig3 has been implicated as a component of a NER subpathway that operates in dividing cells, but which becomes particularly important in nondividing cells. Here, we use DT40 cells and powerful gene targeting approaches for generating DNA ligase mutants to examine the involvement and contribution of Lig1 and Lig3 in NER using cell survival measured by colony formation, and repair kinetics of CPD by immunofluorescence microscopy and immuno‐slot‐blotting. Our results demonstrate an impressive and previously undocumented potential of Lig3 to substitute for Lig1 in removing helix‐distorting DNA lesions by NER in proliferating cells. We show for the first time in a clean genetic background a functional redundancy in NER between Lig1 and Lig3, which appears to be cell cycle independent and which is likely to contribute to the stability of vertebrate genomes.     

An Ethenoadenine FAD Analog Accelerates UV Dimer Repair by DNA Photolyase

Reduced anionic flavin adenine dinucleotide (FADH^?) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV‐damaged DNA. The initial step involves photoinduced electron transfer from *FADH^? to the CPD. The adenine (Ade) moiety is nearly stacked with the flavin ring, an unusual conformation compared to other FAD‐dependent proteins. The role of this proximity has not been unequivocally elucidated. Some studies suggest that Ade is a radical intermediate, but others conclude that Ade modulates the electron transfer rate constant (k_ET) through superexchange. No study has succeeded in removing or modifying this Ade to test these hypotheses. Here, FAD analogs containing either an ethano‐ or etheno‐bridged Ade between the AN1 and AN6 atoms (e‐FAD and ε‐FAD, respectively) were used to reconstitute apo‐PL, giving e‐PL and ε‐PL respectively. The reconstitution yield of e‐PL was very poor, suggesting that the hydrophobicity of the ethano group prevented its uptake, while ε‐PL showed 50% reconstitution yield. The substrate binding constants for ε‐PL and rPL were identical. ε‐PL showed a 15% higher steady‐state repair yield compared to FAD‐reconstituted photolyase (rPL). The acceleration of repair in ε‐PL is discussed in terms of an ε‐Ade radical intermediate vs superexchange mechanism.     

PHOTOREACTIVATION IN A phrB MUTANT OF Escherichia coli K-12: EVIDENCE FOR THE ROLE OF A SECOND PROTEIN IN PHOTOREPAIR

Abstract In Escherichia coli , the light-dependent repair of pyrimidine dimers in UV-irradiated DNA is now accepted as being due to enzymatic photoreactivation (PR) by a 50 kDa enzyme, photolyase (EC 4.1.99.3). The gene for this enzyme has been mapped at 16.2 min and designated phr . This gene was earlier described as phr B, another locus phr A having been proposed in association with PR. The relevance of the putative phr A gene has now been placed in doubt. The recent report of the discovery of a photoreactivating enzyme in Drosphila melanogaster . which specifically repairs pyrimidine (6–4) pyrimidone photoproducts ([6–4] photoproducts), and that E. coli does possess a protein with specific affinity for the (6–4) photoproduct, has cast new light on the prospective role of phr A in PR. We have determined the nucleotide sequence of the putative phr A gene, which suggests it codes for a protein of 38 kDa. When the putative phr A gene was cloned into an expression vector and transformed into a phr A phr B mutant of E. coli , a level of photorepair was observed, which could correspond to repair of (6–4) photoproducts.     

UV INDUCED (6-4) PHOTOPRODUCTS ARE DISTRIBUTED DIFFERENTLY THAN CYCLOBUTANE DIMERS IN NUCLEOSOMES

We have compared the distributions of two stable UV photoproducts in nucleosome core DNA at the single-nucleotide level using a T4 polymerase-exonuclease mapping procedure. The distribution of pyrimidine-pyrimidone (6-4) dimers was uncovered by reversing the major UV photo-product, cis-syn cyclobutane pyrimidine dimer, with E. coli DNA photolyase and photoreactivating light. Whereas the distribution of total UV photoproducts in nucleosome core DNA forms a striking 10.3 base periodic pattern, the distribution of (6-4) dimers is much more random throughout the nucleosome core domain. Therefore, histone-DNA interactions in nucleosomes strongly modulate formation of the major class of UV-induced photoproducts, while having either a constant effect or no effect on (6-4) dimer formation.     

A Review of Spectroscopic and Biophysical‐Chemical Studies of the Complex of Cyclobutane Pyrimidine Dimer Photolyase and Cryptochrome DASH with Substrate DNA

Cyclobutane pyrimidine dimer (CPD) photolyase (PL) is a structure‐specific DNA repair enzyme that uses blue light to repair CPD on DNA. Cryptochrome (CRY) DASH enzymes use blue light for the repair of CPD lesions on single‐stranded (ss) DNA, although some may also repair these lesions on double‐stranded (ds) DNA. In addition, CRY DASH may be involved in blue light signaling, similar to cryptochromes. The focus of this review is on spectroscopic and biophysical‐chemical experiments of the enzyme–substrate complex that have contributed to a more detailed understanding of all the aspects of the CPD repair mechanism of CPD photolyase and CRY DASH. This will be performed in the backdrop of the available X‐ray crystal structures of these enzymes bound to a CPD‐like lesion. These structures helped to confirm conclusions that were drawn earlier from spectroscopic and biophysical‐chemical experiments, and they have a critical function as a framework to design new experiments and to interpret new experimental data. This review will show the important synergy between X‐ray crystallography and spectroscopic/biophysical‐chemical investigations that is essential to obtain a sufficiently detailed picture of the overall mechanism of CPD photolyases and CRY DASH proteins.     

Electrochemical approach to the repair of oxetanes mimicking DNA (6-4) photoproducts

Electrochemical study of oxetanes mimicking DNA (6-4) photoproducts gives new insight into the repair mechanism by (6-4) photolyase. Both electrochemical oxidation and electrochemical reduction at carbon electrodes lead to the cleavage of the oxetanes in a retro-Paterno-Büchi sequence. Within the family of compounds investigated and the range of driving forces offered, transient formation of unstable radical ions is observed, for both oxidative and reductive cleavage. Taking advantage of the electrochemical signature of these mimics, enzymatic assay with Escherichia coli CPD photolyase coupled to electrochemical monitoring of the reaction brings evidence that enzymatic repair of (6-4) DNA photoproducts does involve a catalytic dissociative electron-transfer mechanism at the level of an oxetane intermediate.     

UVB-lnduced DNA Damage and Its Photorepair in Nuclei and Chloroplasts of Spinacia oleracea L.

Ultraviolet-B-induced lesions and their photorepair in nuclear and chloroplast DNA of spinach (Spinacia oleracea L.) leaves were examined with two photoproducts, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidinone photoproducts (6-4PP). These photoproducts were induced both in nuclear and chloroplast DNA by UVB irradiation and could be detected by enzyme-linked immunosorbent assay using their respective monoclonal antibodies. Formation of CPD was greater in nuclear DNA than in chloroplast DNA (about 10 to 7), whereas 6-4PP formation was comparable in both DNA. On subsequent exposure of leaves to blue/UVA after UVB irradiation, photorepair of CPD and 6-4PP occurred in nuclear DNA but not in chloroplast DNA. When isolated chloroplasts were irradiated with UVB, CPD was also induced in their DNA. But photorepair of CPD did not occur in them by subsequent exposure to blue/UVA, suggesting that no photorepair system operates in chloroplasts.     

Divalent Cations Increase DNA Repair Activities of Bacterial (6‐4) Photolyases

The (6‐4) photolyases of the FeS‐BCP group can be considered as the most ancient type among the large family of cryptochrome and photolyase flavoproteins. In contrast to other photolyases, they contain an Fe‐S cluster of unknown function, a DMRL chromophore, an interdomain loop, which could interact with DNA, and a long C‐terminal extension. We compared DNA repair and photoreduction of two members of the FeS‐BCP family, Agrobacterium fabrum PhrB and Rhodobacter sphaeroides RsCryB, with a eukaryotic (6‐4) photolyase from Ostreococcus, OsCPF, and a member of the class III CPD photolyases, PhrA from A. fabrum. We found that the low DNA repair effectivity of FeS‐BCP proteins is largely stimulated by Mg²⁺ and other divalent cations, whereas no effect of divalent cations was observed in OsCPF and PhrA. The (6‐4) repair activity in the presence of Mg²⁺ is comparable with the repair activities of the other two photolyases. The photoreduction, on the other hand, is negatively affected by Mg²⁺ in PhrB, but stimulated by Mg²⁺ in PhrA. A clear relationship of Mg²⁺ dependency on DNA repair with the evolutionary position conflicts with Mg²⁺ dependency of photoreduction. We discuss the Mg²⁺ effect in the context of structural data and DNA binding.