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Bioorganic & Medicinal Chemistry Jan 2018Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be...
Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be used to visualize the recruitment of DNA repair proteins at double-strand break (DSB) sites. Interestingly, Hoechst 33258 was used under micro-irradiation to sensitize 5-bromouracil (U)-labelled DNA, causing efficient DSBs. However, the molecular basis of DSB formation under UVA micro-irradiation remains unknown. Herein, we investigated the mechanism of DSB formation under UVA micro-irradiation conditions. Our results suggest that the generation of a uracil-5-yl radical through electron transfer from Hoechst 33258 to U caused DNA cleavage preferentially at self-complementary 5'-AAUU-3' sequences to induce DSB. We also investigated the DNA cleavage in the context of the nucleosome to gain a better understanding of UVA micro-irradiation in a cell-like model. We found that DNA cleavage occurred in both core and linker DNA regions although its efficiency reduced in core DNA.
Topics: Bisbenzimidazole; Bromouracil; DNA; DNA Breaks, Double-Stranded; DNA Cleavage; Dose-Response Relationship, Drug; Free Radicals; Molecular Structure; Structure-Activity Relationship; Ultraviolet Rays
PubMed: 29170027
DOI: 10.1016/j.bmc.2017.11.011 -
The Journal of Physical Chemistry. A Jun 2017Halogen-substituted pyrimidines, such as 5-bromouracil and 5-iodouracil, have been used as radio therapeutic (RT) agents in cancer treatment. The radiosensitizing...
Halogen-substituted pyrimidines, such as 5-bromouracil and 5-iodouracil, have been used as radio therapeutic (RT) agents in cancer treatment. The radiosensitizing activity of 5-bromouracil is attributed to its reaction with electron which produce the highly reactive uracil-5-yl radical by dissociating the C5-Br bond. Using density functional methods and highly accurate Gaussian 4 (G4) theory, herein, we show that 5-bromocytosine (5-Brcyt) after reaction with electron, also, leads to the formation of cytosine-5-yl radical. However, our results show that this species can subsequently undergo a base-catalyzed tautomerization reaction to form the π-aminyl radical followed by a second tautomerization to the thermodynamically most stable σ-iminyl radical (cytN). From the present theoretical calculations, we infer that the mechanism of the formation of cytN by one-electron reduction of 5-Brcyt is straightforward and may take place in 5-Brcyt-labeled DNA in competition with the usual reactions expected for the cytosine-5-yl radical such as abstraction and water addition.
PubMed: 28586202
DOI: 10.1021/acs.jpca.7b04034 -
Cell Cycle (Georgetown, Tex.) Feb 2017Transcriptional timing is inherently influenced by gene length, thus providing a mechanism for temporal regulation of gene expression. While gene size has been shown to...
Transcriptional timing is inherently influenced by gene length, thus providing a mechanism for temporal regulation of gene expression. While gene size has been shown to be important for the expression timing of specific genes during early development, whether it plays a role in the timing of other global gene expression programs has not been extensively explored. Here, we investigate the role of gene length during the early transcriptional response of human fibroblasts to serum stimulation. Using the nascent sequencing techniques Bru-seq and BruUV-seq, we identified immediate genome-wide transcriptional changes following serum stimulation that were linked to rapid activation of enhancer elements. We identified 873 significantly induced and 209 significantly repressed genes. Variations in gene size allowed for a large group of genes to be simultaneously activated but produce full-length RNAs at different times. The median length of the group of serum-induced genes was significantly larger than the median length of all expressed genes, housekeeping genes, and serum-repressed genes. These gene length relationships were also observed in corresponding mouse orthologs, suggesting that relative gene size is evolutionarily conserved. The sizes of transcription factor and microRNA genes immediately induced after serum stimulation varied dramatically, setting up a cascade mechanism for temporal expression arising from a single activation event. The retention and expansion of large intronic sequences during evolution have likely played important roles in fine-tuning the temporal expression of target genes in various cellular response programs.
Topics: Animals; Bromouracil; Conserved Sequence; Enhancer Elements, Genetic; Evolution, Molecular; Fibroblasts; Gene Expression Regulation; Genes; Humans; Male; Mice; MicroRNAs; Models, Biological; Oligonucleotide Array Sequence Analysis; Serum; Serum Response Factor; Time Factors; Transcription Factors; Transcription, Genetic; Uridine
PubMed: 28055303
DOI: 10.1080/15384101.2016.1234550 -
Nucleic Acids Research Dec 2014In a previous study, we found that 2-deoxyribonolactone is effectively generated in the specific 5-bromouracil ((Br)U)-substituted sequence 5'-(G/C)[A]n = 1,2...
In a previous study, we found that 2-deoxyribonolactone is effectively generated in the specific 5-bromouracil ((Br)U)-substituted sequence 5'-(G/C)[A]n = 1,2 (Br)U(Br)U-3' and proposed that a formed uracil-5-yl radical mainly abstracts the C1' hydrogen from the 5'-side of (Br)U(Br)U under 302-nm irradiation condition. In the present work, we performed photoirradiation of (Br)U-substituted DNA in the presence of a hydrogen donor, tetrahydrofuran, to quench the uracil-5-yl radical to uracil and then subjected the sample to uracil DNA glycosylase digestion. Slab gel sequence analysis indicated that uracil residues were formed at the hot-spot sequence of 5'-(G/C)[A]n = 1,2 (Br)U(Br)U-3' in 302-nm irradiation of (Br)U-substituted DNA. Furthermore, we found that the uracil residue was also formed at the reverse sequence 5'-(Br)U(Br)U[A]n = 1,2(G/C)-3', which suggests that both 5'-(G/C)[A]n = 1,2 (Br)U(Br)U-3' and 5'-(Br)U(Br)U[A]n = 1,2(G/C)-3' are hot-spot sequences for the formation of the uracil-5-yl radical.
Topics: Bromouracil; DNA; Electrophoresis, Polyacrylamide Gel; Furans; Sequence Analysis, DNA; Ultraviolet Rays; Uracil; Uracil-DNA Glycosidase
PubMed: 25398904
DOI: 10.1093/nar/gku1133 -
Nucleic Acids Research Aug 2016Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including...
Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein-DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, (Br)U-labelled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction.
Topics: Base Sequence; Bromouracil; DNA; DNA Cleavage; Electrons; Enhancer Elements, Genetic; Humans; Hypoxanthine; Light; PAX6 Transcription Factor; Protein Binding; Protein Domains; Protein Structure, Secondary; Reproducibility of Results; SOXB1 Transcription Factors; Spectrometry, Fluorescence; Structure-Activity Relationship; Tryptophan; Uridine
PubMed: 27229137
DOI: 10.1093/nar/gkw478 -
Chemical Communications (Cambridge,... Oct 2015We report the photochemistry of (Br)U substituted DNA as a versatile platform to investigate the binding sites of pyrene conjugated pyrrole imidazole polyamides (PIPs)....
We report the photochemistry of (Br)U substituted DNA as a versatile platform to investigate the binding sites of pyrene conjugated pyrrole imidazole polyamides (PIPs). The results suggest that the approach can be used on a routine basis for the screening of polyamide binding sites.
Topics: Binding Sites; Bromouracil; DNA; Electrons; Nylons
PubMed: 26278406
DOI: 10.1039/c5cc05104e