Trioxsalen

Systematic analysis of DNA crosslink repair pathways during development and aging in Caenorhabditis elegans.[Pubmed:28934497]

Nucleic Acids Res. 2017 Sep 19;45(16):9467-9480.

DNA interstrand crosslinks (ICLs) are generated by endogenous sources and chemotherapeutics, and pose a threat to genome stability and cell survival. Using Caenorhabditis elegans mutants, we identify DNA repair factors that protect against the genotoxicity of ICLs generated by Trioxsalen/ultraviolet A (TMP/UVA) during development and aging. Mutations in nucleotide excision repair (NER) components (e.g. XPA-1 and XPF-1) imparted extreme sensitivity to TMP/UVA relative to wild-type animals, manifested as developmental arrest, defects in adult tissue morphology and functionality, and shortened lifespan. Compensatory roles for global-genome (XPC-1) and transcription-coupled (CSB-1) NER in ICL sensing were exposed. The analysis also revealed contributions of homologous recombination (BRC-1/BRCA1), the MUS-81, EXO-1, SLX-1 and FAN-1 nucleases, and the DOG-1 (FANCJ) helicase in ICL resolution, influenced by the replicative-status of the cell/tissue. No obvious or critical role in ICL repair was seen for non-homologous end-joining (cku-80) or base excision repair (nth-1, exo-3), the Fanconi-related proteins BRC-2 (BRCA2/FANCD1) and FCD-2 (FANCD2), the WRN-1 or HIM-6 (BLM) helicases, or the GEN-1 or MRT-1 (SNM1) nucleases. Our efforts uncover replication-dependent and -independent ICL repair networks, and establish nematodes as a model for investigating the repair and consequences of DNA crosslinks in metazoan development and in adult post-mitotic and proliferative germ cells.

Elements That Regulate the DNA Damage Response of Proteins Defective in Cockayne Syndrome.[Pubmed:26616585]

J Mol Biol. 2016 Jan 16;428(1):62-78.

Cockayne syndrome (CS) is a premature aging disorder characterized by developmental defects, multisystem progressive degeneration and sensitivity to ultraviolet light. CS is divided into two primary complementation groups, A and B, with the CSA and CSB proteins presumably functioning in DNA repair and transcription. Using laser microirradiation and confocal microscopy, we characterized the nature and regulation of the CS protein response to oxidative DNA damage, double-strand breaks (DSBs), angelicin monoadducts and Trioxsalen interstrand crosslinks (ICLs). Our data indicate that CSB recruitment is influenced by the type of DNA damage and is most rapid and robust as follows: ICLs>DSBs>monoadducts>oxidative lesions. Transcription inhibition reduced accumulation of CSB at sites of monoadducts and ICLs, but it did not affect recruitment to (although slightly affected retention at) oxidative damage. Inhibition of histone deacetylation altered the dynamics of CSB assembly, suggesting a role for chromatin status in the response to DNA damage, whereas the proteasome inhibitor MG132 had no effect. The C-terminus of CSB and, in particular, its ubiquitin-binding domain were critical to recruitment, while the N-terminus and a functional ATPase domain played a minor role at best in facilitating protein accumulation. Although the absence of CSA had no effect on CSB recruitment, CSA itself localized at sites of ICLs, DSBs and monoadducts but not at oxidative lesions. Our results reveal molecular components of the CS protein response and point to a major involvement of complex lesions in the pathology of CS.

CSB interacts with SNM1A and promotes DNA interstrand crosslink processing.[Pubmed:25505141]

Nucleic Acids Res. 2015 Jan;43(1):247-58.

Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5'-3' exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized Trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and gamma-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

Detecting RNA-RNA interactions using psoralen derivatives.[Pubmed:25183819]

Cold Spring Harb Protoc. 2014 Sep 2;2014(9):996-1000.

Psoralens are tricyclic compounds that intercalate into double-stranded DNA or RNA and, on irradiation with long-wavelength (365-nm) UV light, covalently link pyrimidines on adjacent strands. More rarely, psoralen cross-links can be observed at the ends of helices (i.e., double-stranded-single-stranded boundaries). Although psoralens can, in some instances, cross-link protein to RNA, their primary application is to detect RNA-RNA base-pairing interactions. The most useful psoralen derivative is 4'-aminomethyl Trioxsalen (AMT), which is soluble in H2O. This protocol describes the use of AMT to detect RNA-RNA interactions in tissue culture cells or in extracts. Cross-linked RNAs are detectable by their reduced mobility in polyacrylamide gels. Cross-links can be reversed by exposure to short-wavelength (254 nm) UV light.

[Simultaneous determination of eight furocoumarines in cosmetics by high performance liquid chromatography and verification by liquid chromatography-tandem mass spectrometry].[Pubmed:24010339]

Se Pu. 2013 May;31(5):416-22.

A method using high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of eight furocoumarines (8-hydroxypsoralen, psoralen, isopsoralen, 8-methoxypsoralen, 5-methoxypsoralen, Trioxsalen, imperatorin and isoimperatorin) in cosmetics. The cosmetic samples, including cream, lotion, shampoo, powder and lipstick, were supersonically extracted with appropriate solvents. The extract was centrifuged, and the supernatant was filtered through a membrane, and then separated on an Agilent Zorbax SB-Phenyl chromatographic column (250 mm x 4.6 mm, 5 microm) by gradient elution at a flow rate of 1.0 mL/min with methanol-acetonitrile-water as mobile phases. The column temperature was set at 30 degrees C. The wavelength of detection was 250 nm. The limits of quantification (LOQs) were 0.25 mg/kg for 8-hydroxypsoralen and 0.5 mg/kg for psoralen, isopsoralen, 8-methoxypsoralen, 5-methoxypsoralen, Trioxsalen, imperatorin and isoimperatorin. The recoveries at three spiked levels were in the range of 85.0% - 105.8% with the relative standard deviations (RSDs) of 0.41% - 7.90%. The intra-day precision (n=6) was less than 1%, and the inter-day precision (n = 6) was less than 2% for the peak areas of the eight furocoumarines in a mixed standard solution. The method is accurate, simple, rapid and suitable for the determination of the eight furocoumarines in various cosmetic samples.

Dengue virus photo-inactivated in presence of 1,5-iodonaphthylazide (INA) or AMT, a psoralen compound (4'-aminomethyl-trioxsalen) is highly immunogenic in mice.[Pubmed:23835446]

Hum Vaccin Immunother. 2013 Nov;9(11):2336-41.

Two novel methods of dengue virus inactivation using iodonaphthyl azide (INA) and aminomethyl Trioxsalen (AMT) were compared with traditional virus inactivation by formaldehyde. The AMT inactivated dengue-2 virus retained its binding to a panel of 5 monoclonal antibodies specific for dengue-2 envelope protein, whereas inactivation by formaldehyde and INA led to 30-50% decrease in binding. All three inactivated viruses elicited high level virus neutralizing antibodies in vaccinated mice. However, only mice vaccinated with AMT inactivated virus mounted T cell responses similar to live, uninactivated virus.