Repair Of Cyclobutane Pyrimidine Dimers Research Articles

SUMOylation of damaged DNA-binding protein DDB2

Damaged DNA-binding protein (DDB) is a heterodimer composed of two subunits, p127 and p48, which have been designated DDB1 and DDB2, respectively. DDB2 recognizes and binds to UV-damaged DNA during nucleotide excision repair. Here, we demonstrated that DDB2 was SUMOylated in a UV-dependent manner, and its major SUMO E3 ligase was PIASy as determined by RNA interference-mediated knockdown. The UV-induced physical interaction between DDB2 and PIASy supported this notion. PIASy knockdown reduced the removal of cyclobutane pyrimidine dimers (CPDs) from total genomic DNA, but did not affect that of 6-4 pyrimidine pyrimidone photoproducts (6-4PPs). Thus, DDB2 plays an indispensable role in CPD repair, but not in 6-4PP repair, which is consistent with the observation that DDB2 was SUMOylated by PIASy. These results suggest that the SUMOylation of DDB2 facilitates CPD repair.

Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells

Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.

UV-induced Nucleotide Excision Repair (NER) and Photoreactivation Repair (PER) in two trout fish cell lines used in ecotoxicological assessment studies

A better knowledge of DNA repair capacities in permanent fish cell lines would contribute to establish their interest in genotoxicity testing for environmental risk assessment studies including the effects of an increase in solar UV radiations on aquatic organisms. NER (Nucleotide Excision Repair) and PER (Photoreactivation Repair) are the two repair pathways of choice for UV-induced photo-lesions. In the present paper, these repair processes were characterized in the two rainbow trout cell lines, RTGill-W1 and RTL-W1 (liver), by means of a T4-modified comet assay which allowed to follow the cyclobutane pyrimidine dimers repair kinetics specifically. Both repair processes have been found in the cell lines, PER repairing much faster UV lesions than NER, and NER being slightly more efficient in the gill cell line than in the liver one.

Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams

UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UV Tolerant Rice 319), was isolated from a mutagenized population derived from 2500 M1 seeds (of the UVB-resistant cultivar ‘Sasanishiki’) that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, utr319 is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined.

UVB‐Induced Inflammatory Cytokine Release, DNA Damage and Apoptosis of Human Oral Compared with Skin Tissue Equivalents

People can get oral cancers from UV (290-400 nm) exposures. Besides high outdoor UV exposures, high indoor UV exposures to oral tissues can occur when consumers use UV-emitting tanning devices to either tan or whiten their teeth. We compared the carcinogenic risks of skin to oral tissue cells after UVB (290-320 nm) exposures using commercially available 3D-engineered models for human skin (EpiDerm™), gingival (EpiGing™) and oral (EpiOral™) tissues. To compare the relative carcinogenic risks, we investigated the release of cytokines, initial DNA damage in the form of cyclobutane pyrimidine dimers (CPDs), repair of CPDs and apoptotic cell numbers. We measured cytokine release using cytometric beads with flow cytometry and previously developed a fluorescent immunohistochemical assay to quantify simultaneously CPD repair rates and apoptotic cell numbers. We found that interleukin-8 (IL-8) release and the initial CPDs are significantly higher, whereas the CPD repair rates and apoptotic cell numbers are significantly lower for oral compared with skin tissue cells. Thus, the increased release of the inflammatory cytokine IL-8 along with inefficient CPD repair and decreased death rates for oral compared with skin tissue cells suggests that mutations are accumulating in the surviving population of oral cells increasing people's risks for getting oral cancers.

Silibinin inhibits ultraviolet B radiation-induced DNA-damage and apoptosis by enhancing interleukin-12 expression in JB6 cells and SKH-1 hairless mouse skin

Recent studies have demonstrated silibinin efficacy against ultraviolet B (UVB)-induced skin carcinogenesis via different mechanisms in cell lines and animal models; however, its role in regulating interleukin-12 (IL-12), an immunomodulatory cytokine that reduces UVB-induced DNA damage and apoptosis, is not known. Here, we report that UVB irradiation causes caspase 3 and PARP cleavage and apoptosis, and addition of recombinant IL-12 or silibinin immediately after UVB significantly protects UVB-induced apoptosis in JB6 cells. IL-12 antibody-mediated blocking of IL-12 activity compromised the protective effects of both IL-12 and silibinin. Both silibinin and IL-12 also accelerated the repair of UVB-caused cyclobutane-pyrimidine dimers (CPDs) in JB6 cells. Additional studies confirmed that indeed silibinin causes a significant increase in IL-12 levels in UVB-irradiated JB6 cells as well as in mouse skin epidermis, and that similar to cell-culture findings, silibinin topical application immediately after UVB exposure causes a strong protection against UVB-induced TUNEL positive cells in epidermis possibly through a significantly accelerated repair of UVB-caused CPDs. Together, these findings for the first time provide an important insight regarding the pharmacological mechanism wherein silibinin induces endogenous IL-12 in its efficacy against UVB-caused skin damages. In view of the fact that an enhanced endogenous IL-12 level could effectively remove UVB-caused DNA damage and associated skin cancer, our findings suggest that the use of silibinin in UVB-damaged human skin would also be a practical and translational strategy to manage solar radiation-caused skin damages as well as skin cancer.

How does the "ancient" asexual Philodina roseola (Rotifera: Bdelloidea) handle potential UVB-induced mutations?

Like other obligate asexuals, bdelloid rotifers are expected to suffer from degradation of their genomes through processes including the accumulation of deleterious mutations. However, sequence-based analyses in this regard remain inconclusive. Instead of looking for historical footprints of mutations in these ancient asexuals, we directly examined the susceptibility and ability to repair point mutations by the bdelloid Philodina roseola by inducing cyclobutane-pyrimidine dimers (CPDs) via exposure to UVB radiation (280-320 nm). For comparison, we performed analogous experiments with the facultative asexual monogonont rotifer Brachionus rubens. Different strategies were found for the two species. Philodina roseola appeared to shield itself from CPD induction through uncharacterized UV-absorbing compounds and, except for the genome reconstruction that occurs after desiccation, was largely unable to repair UVB-induced damage. By contrast, B. rubens was more susceptible to UVB irradiation, but could repair all induced damage in ~2 h. In addition, whereas UV irradiation had a significant negative impact on the reproductive output of P. roseola, and especially so after desiccation, that of B. rubens was unaffected. Although the strategy of P. roseola might suffice under natural conditions where UVB irradiation is less intense, the lack of any immediate CPD repair mechanisms in this species remains perplexing. It remains to be investigated how typical these results are for bdelloids as a group and therefore how reliant these animals are on desiccation-dependent genome repair to correct potential DNA damage given their obligate asexual lifestyle.

HCMV-Infected Cells Maintain Efficient Nucleotide Excision Repair of the Viral Genome while Abrogating Repair of the Host Genome

Many viruses subvert the host cell's ability to mount and complete various DNA damage responses (DDRs) after infection. HCMV infection of permissive fibroblasts activates host DDRs at the time of viral deposition and during replication, but the DDRs remain uncompleted without arrest or apoptosis. We believe this was in part due to partitioning of the damage response and double strand break repair components. After extraction of soluble proteins, the localization of these components fell into three groups: specifically associated with the viral replication centers (RCs), diffused throughout the nucleoplasm and excluded from the RCs. Others have shown that cells are incapable of processing exogenously introduced damage after infection. We hypothesized that the inability of the cells to process damage might be due to the differential association of repair components within the RCs and, in turn, potentially preferential repair of the viral genome and compromised repair of the host genome. To test this hypothesis we used multiple strategies to examine repair of UV-induced DNA damage in mock and virus-infected fibroblasts. Comet assays indicated that repair was initiated, but was not completed in infected cells. Quantitative analysis of immunofluorescent localization of cyclobutane pyrimidine dimers (CPDs) revealed that after 24 h of repair, CPDs were significantly reduced in viral DNA, but not significantly changed in the infected host DNA. To further quantitate CPD repair, we developed a novel dual-color Southern protocol allowing visualization of host and viral DNA simultaneously. Combining this Southern methodology with a CPD-specific T4 endonuclease V alkaline agarose assay to quantitate repair of adducts, we found efficient repair of CPDs from the viral DNA but not host cellular DNA. Our data confirm that NER functions in HCMV-infected cells and almost exclusively repairs the viral genome to the detriment of the host's genome.

Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA

Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA−/− cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6–4) photoproducts, and are consequently more arrested in S phase as compared to XPA−/− cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.

Abstract A85: Silibinin exerts its protective effects against UVB-induced photodamage and skin carcinogenesis through p53

Abstract Ultraviolet (UV)-caused photodamage is the primary etiological factor for non-melanoma skin cancers (NMSCs), the most common malignancy in the United States. Better preventive strategies are urgently required to reduce UV-caused photodamage as well as the incidence of NMSCs. The tumor suppressor gene p53 is considered the most important cellular transcriptional factor for preserving genomic stability, and plays a major protective role against UVB-induced photodamage in epidermal cells. Mutated p53 patches are an early molecular biomarker and considered one of the most important characteristics of UVB-induced skin carcinogenesis. Earlier, in vitro and in vivo studies have shown that the natural flavonolignan silibinin activates p53 as well as reduces photodamage and NMSCs; but whether silibinin channels its protective effects primarily through p53 remains unknown. Herein, we generated p53 heterozygous (p53+/−) and p53 null (p53−/−) mice on SKH-1 hairless mouse background to investigate this issue. In wild-type SKH-1 mice, topical application of silibinin (9 mg/200 μl acetone) pre- or post- UVB (180 mJ/cm2) irradiation significantly reduced the formation of CPDs (cyclobutane pyrimidine dimers) after 12 h, while CPD repair by silibinin was significantly compromised in p53−/− mice. In the skin carcinogenesis study, wild type (p53+/+) and heterozygous (p53+/-) SKH-1 hairless mice were exposed to UVB (90 mJ/cm2, 5 times per week), while a subset was administered silibinin topically (9 mg/200 μl acetone) pre- or post-UVB irradiation. Mice were sacrificed at various time-points (4, 8, and 25 weeks) and skin samples were collected and analyzed. Analyses for proliferation marker (PCNA) in the early time-points (4 and 8 weeks) showed that the proliferation rate was higher in p53+/- mice compared to p53+/+ mice; furthermore, the strong inhibitory effect of silibinin on PCNA was evident in p53+/+ mice but compromised in p53+/- mice. At the end of the 25 weeks, p53+/- mice developed skin tumors earlier than p53+/+ mice; and p53+/- mice showed higher tumor number, multiplicity, and volume as compared to p53+/+ mice. Results also showed that silibinin treatment significantly reduced the tumor number, multiplicity and volume in p53+/+ mice but its protective efficacy was significantly compromised in p53+/- mice. In the case of p53+/+ mice, silibinin treatment (pre- or post-UVB) significantly reduced tumor volume in irradiated animals (5.3 and 18.6 mm3/per mouse compared to 109.5 mm3/per mouse in UVB-alone irradiated group; P<0.001) accounting for 95% and 83% reduction in tumor volume respectively. While in p53+/- mice, UVB irradiation induced significantly larger tumors (503.7 mm3), in the case of silibinin pre- and post-treatment the tumor volume was reduced to 293 mm3 (P<0.05 versus UVB-alone) and 425 mm3 accounting for only 42% and 15% reduction respectively. These results not only reestablished previous observations that absence of p53 allele significantly predisposes animals to photodamage and photocarcinogenesis, but also showed for the first time that silibinin mediates its protection against UVB-induced photodamage and photocarcinogenesis in a p53-dependent manner. These mechanistic studies support the use of silibinin as chemopreventive agent in NMSCs. Citation Format: Cynthia Tilley, Srirupa Roy, Gagan Deep, Chapla Agarwal, Rajesh Agarwal. Silibinin exerts its protective effects against UVB-induced photodamage and skin carcinogenesis through p53. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A85.

Nucleotide Excision Repair Is Reduced in Oral Epithelial Tissues Compared with Skin‡

Ultraviolet radiation (UVR) exposure to internal tissues for diagnostic, therapeutic and cosmetic procedures has increased dramatically over the past decade. The greatest increase in UVR exposure of internal tissues occurs in the cosmetic industry where it is combined with oxidizing agents for teeth whitening, often in conjunction with indoor tanning. To address potential carcinogenic risks of these procedures, we analyzed the formation and repair of the DNA photoproducts associated with the signature mutations of UVR. Radioimmunoassay was used to quantify the induction and repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts in DNA purified from three reconstructed tissues, EpiDerm(TM) , EpiGingival(TM) and EpiOral(TM) . We observed comparable levels of DNA damage in all tissues immediately after UVR exposure. In contrast, repair was significantly reduced in both oral tissues compared with EpiDerm(TM) . Our data suggest that UVR exposure of oral tissues can result in accumulation of DNA damage and increase the risk for carcinoma and melanoma of the mouth. Because NER is a broad-spectrum defense against DNA damage caused by a variety of agents in addition to UVR, our data suggest that the relatively low NER efficiency observed in oral tissues may have wide-ranging consequences in this highly exposed environment.

Electron Tunneling Pathways and Role of Adenine in Repair of Cyclobutane Pyrimidine Dimer by DNA Photolyase

Electron tunneling pathways in enzymes are critical to their catalytic efficiency. Through electron tunneling, photolyase, a photoenzyme, splits UV-induced cyclobutane pyrimidine dimer into two normal bases. Here, we report our systematic characterization and analyses of photoinitiated three electron transfer processes and cyclobutane ring splitting by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. We observed the complete dynamics of the reactants, all intermediates and final products, and determined their reaction time scales. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repair and for the final electron return to restore the active cofactor and complete the catalytic photocycle. Significantly, we found that the adenine moiety of the unusual bent flavin cofactor is essential to mediating all electron-transfer dynamics through a superexchange mechanism, leading to a delicate balance of time scales. The cyclobutane ring splitting takes tens of picoseconds, while electron-transfer dynamics all occur on a longer time scale. The active-site structural integrity, unique electron tunneling pathways, and the critical role of adenine ensure the synergy of these elementary steps in this complex photorepair machinery to achieve maximum repair efficiency which is close to unity. Finally, we used the Marcus electron-transfer theory to evaluate all three electron-transfer processes and thus obtained their reaction driving forces (free energies), reorganization energies, and electronic coupling constants, concluding that the forward and futile back-electron transfer is in the normal region and that the final electron return of the catalytic cycle is in the inverted region.

Abstract 1592: Silibinin inhibits UVB-induced DNA-damage and apoptosis by enhancing interleukin-12 expression in JB6 cells and SKH-1 mouse skin

Abstract Ultraviolet-B (UVB) radiation plays a pivotal role in non-melanoma human skin cancers (NMSCs) by inducing cellular DNA damage, mutations and immunosuppression. Decades of research has demonstrated silibinin efficacy against UVB-induced skin carcinogenesis by regulating different molecular pathways in various cell lines and animal models; however, the role of silibinin in regulating interleukin-12 (IL-12), an immunomodulatory cytokine with the capacity to reduce UVB-induced DNA damage and apoptosis, has not been studied. Here, we report that UVB irradiation caused a dose- and time-dependent caspase 3 and PARP cleavage as well as apoptosis in JB6 cells. Addition of recombinant IL-12 or silibinin immediately after UVB significantly protected UVB-induced apoptosis in JB6 cells, and antibody-mediated blocking of IL-12 activity compromised the protective effect of both IL-12 and silibinin. In addition, both silibinin and IL-12 accelerated the repair of UVB-caused cyclobutane-pyrimidine dimers (CPDs) in JB6 cells. Additional studies confirmed that indeed silibinin causes a significant increase in IL-12 levels in UVB-irradiated JB6 cells as well as in the SKH-1 hairless mouse epidermis, and that similar to in vitro findings, silibinin topical application immediately after UVB exposure causes a very strong protection against UVB-induced TUNEL positive cells in epidermis possibly through a significantly accelerated repair of UVB-caused CPDs in skin epidermis. Together, these findings for the first time identify an important insight regarding the pharmacological mechanism wherein silibinin induces endogenous IL-12 in its efficacy against UVB-caused skin damages. Considering the fact that millions of people get constantly exposed to solar UVB radiation and live with persistent UVB-induced DNA lesions, the post-damage use of silibinin as an inducer of endogenous IL-12 in UVB-damaged human skin for the rapid repair of DNA damage could be a more practical and translational approach in reducing sunlight-caused damages in human skin which eventually lead to NMSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1592. doi:1538-7445.AM2012-1592

Fully functional global genome repair of (6-4) photoproducts and compromised transcription-coupled repair of cyclobutane pyrimidine dimers in condensed mitotic chromatin

During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression.

Electron Tunneling Pathways and Role of Adenine in Repair of Cyclobutane Pyrimidine Dimer by DNA Photolyase

Electron Tunneling Pathways and Role of Adenine in Repair of Cyclobutane Pyrimidine Dimer by DNA Photolyase

Silibinin prevents ultraviolet B radiation-induced epidermal damages in JB6 cells and mouse skin in a p53-GADD45α-dependent manner

Better preventive strategies are required to reduce ultraviolet (UV)-caused photodamage, the primary etiological factor for non-melanoma skin cancer (NMSC). Accordingly, here we examined the preventive efficacy of silibinin against UVB-induced photodamage using mouse epidermal JB6 cells and SKH1 hairless mouse epidermis. In JB6 cells, silibinin pretreatment protected against apoptosis and accelerated the repair of cyclobutane pyrimidine dimers (CPD) induced by moderate dose of UVB (50 mJ/cm(2)), which we are at risk of daily exposure. Silibinin also reversed UVB-induced S phase arrest, reducing both active DNA synthesizing and inactive S phase populations. In mechanistic studies, UVB-irradiated cells showed a transient upregulation of both phosphorylated (Ser-15 and Ser-392) and total p53, whereas silibinin pretreatment led to a more sustained upregulation and stronger nuclear localization of p53. Silibinin also caused a marked upregulation of GADD45α, a downstream target of p53, implicated in DNA repair and cell cycle regulation. Importantly, under p53 and GADD45α knockdown conditions, cells were more susceptible to UVB-induced apoptosis without any significant S phase arrest, and protective effects of silibinin were compromised. Similar to the in vitro results, topical application of silibinin prior to or immediately after UVB irradiation resulted in sustained increase in p53 and GADD45α levels and accelerated CPD removal in the epidermis of SKH1 hairless mice. Together, our results show for the first time that p53-mediated GADD45α upregulation is the key mechanism by which silibinin protects against UVB-induced photodamage and provides a strong rationale to investigate silibinin in reducing the risk and/or preventing early onset of NMSC.

Structure of full-length Drosophila cryptochrome.

The Cryptochrome/Photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to UV and blue light exposure in all kingdoms of life 1; 2; 3; 4; 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs)and 6-4 photolesions caused by UV radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1; 2; 3; 4; 5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3; 6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalyzed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable C-terminal tail that appends the conserved PL homology domain (PHD) and is important for function 7; 8; 9; 10; 11; 12. Herein, we report a 2.3 Å resolution crystal structure of Drosophila CRY with an intact C-terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic center to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture, and photochemistry can be elaborated into a range of light-driven functions.

Neutral Histidine and Photoinduced Electron Transfer in DNA Photolyases

The two major UV-induced DNA lesions, the cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts, can be repaired by the light-activated enzymes CPD and (6-4) photolyases, respectively. It is a long-standing question how the two classes of photolyases with alike molecular structure are capable of reversing the two chemically different DNA photoproducts. In both photolyases the repair reaction is initiated by photoinduced electron transfer from the hydroquinone-anion part of the flavin adenine dinucleotide (FADH(-)) cofactor to the photoproduct. Here, the state-of-the-art XMCQDPT2-CASSCF approach was employed to compute the excitation spectra of the respective active site models. It is found that protonation of His365 in the presence of the hydroquinone-anion electron donor causes spontaneous, as opposed to photoinduced, coupled proton and electron transfer to the (6-4) photoproduct. The resulting neutralized biradical, containing the neutral semiquinone and the N3'-protonated (6-4) photoproduct neutral radical, corresponds to the lowest energy electronic ground-state minimum. The high electron affinity of the N3'-protonated (6-4) photoproduct underlines this finding. Thus, it is anticipated that the (6-4) photoproduct repair is assisted by His365 in its neutral form, which is in contrast to the repair mechanisms proposed in the literature. The repair via hydroxyl group transfer assisted by neutral His365 is considered. The repair involves the 5'base radical anion of the (6-4) photoproduct which in terms of electronic structure is similar to the CPD radical anion. A unified model of the CPD and (6-4) photoproduct repair is proposed.

Diverse Roles of RNA Polymerase II-associated Factor 1 Complex in Different Subpathways of Nucleotide Excision Repair

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two pathways of nucleotide excision repair (NER). In Saccharomyces cerevisiae, Rad26 is important but not absolutely required for TCR. Rpb4, a nonessential RNA polymerase II (Pol II) subunit that forms a subcomplex with Rpb7, and the Spt4-Spt5 complex, a transcription elongation factor, have been shown to suppress Rad26-independent TCR. The Pol II-associated factor 1 complex (Paf1C) has been shown to function in transcription elongation, 3'-processing of mRNAs, and posttranslational modification of histones. Here we show that Paf1C plays a marginal role in facilitating Rad26-dependent TCR but significantly suppresses Rad26-independent TCR. The suppression of Rad26-independent TCR is achieved by cooperating with Spt4-Spt5. We propose a model that, in the absence of Rad26, a lesion is "locked" in the active center of a Pol II elongation complex, which is stabilized by the coordinated interactions of Rpb4-Rpb7, Spt4-Spt5, and Paf1C with each other and with the core Pol II. We also found that Paf1C facilitates GGR, especially in internucleosomal linker regions. The facilitation of GGR is achieved through enabling monoubiquitination of histone H2B lysine 123 by Bre1, which in turn permits di- and trimethylation of histone H3 lysine 79 by Dot1. To our best knowledge, among the NER-modulating factors documented so far, Paf1C appears to have the most diverse functions in different NER pathways or subpathways.

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase

Photolyase uses blue light to restore the major ultraviolet (UV)-induced DNA damage, the cyclobutane pyrimidine dimer (CPD), to two normal bases by splitting the cyclobutane ring. Our earlier studies showed that the overall repair is completed in 700 ps through a cyclic electron-transfer radical mechanism. However, the two fundamental processes, electron-tunneling pathways and cyclobutane ring splitting, were not resolved. Here, we use ultrafast UV absorption spectroscopy to show that the CPD splits in two sequential steps within 90 ps and the electron tunnels between the cofactor and substrate through a remarkable route with an intervening adenine. Site-directed mutagenesis reveals that the active-site residues are critical to achieving high repair efficiency, a unique electrostatic environment to optimize the redox potentials and local flexibility, and thus balance all catalytic reactions to maximize enzyme activity. These key findings reveal the complete spatio-temporal molecular picture of CPD repair by photolyase and elucidate the underlying molecular mechanism of the enzyme's high repair efficiency.