Investigating Nucleosome Accessibility for MNase, FeII †Peplomycin, and Duocarmycin†B2 by Using Capillary Electrophoresis.

Capillary electrophoresis, coupled with DNA 5' Texas Red labeling, was used to investigate the ability of MNase, FeII peplomycin, and duocarmycin B2 to access the nucleosome. Distinct accessibility patterns of these species to the nucleosome were observed. MNase was completely prevented from approaching the nucleosome core and exhibited a higher site specificity for targeting DNA sites located close to the core region. Intercalation of peplomycin in the nucleosomal core region was highly suppressed, but reaction sites located at the ends of the nucleosomal core remained accessible, which implied flexibility of the core DNA end. Duocarmycin B2 was able to enter and react in the core region, although its alkylating efficiency decreased significantly.

UVA irradiation of BrU-substituted DNA in the presence of Hoechst 33258.

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 (BrU)-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 BrU caused DNA cleavage preferentially at self-complementary 5'-AABrUBrU-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.

Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay.

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.

Identification of Sequence Specificity of 5-Methylcytosine Oxidation by Tet1 Protein with High-Throughput Sequencing.

Tet (ten-eleven translocation) family proteins have the ability to oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic-level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high-throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10(4) -member mC-containing random DNA-sequence library was constructed. The library was subjected to Tet-reactive pulldown followed by high-throughput sequencing. Analysis of the obtained sequence data identified the Tet1-reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein.

Nucleosome Assembly Alters the Accessibility of the Antitumor Agent Duocarmycin B2 to Duplex DNA.

To evaluate the reactivity of antitumor agents in a nucleosome architecture, we conducted in vitro studies to assess the alkylation level of duocarmycin B2 on nucleosomes with core and linker DNA using sequencing gel electrophoresis. Our results suggested that the alkylating efficiencies of duocarmycin B2 were significantly decreased in core DNA and increased at the histone-free linker DNA sites when compared with naked DNA conditions. Our finding that nucleosome assembly alters the accessibility of duocarmycin B2 to duplex DNA could advance its design as an antitumor agent.

Preferential 5-Methylcytosine Oxidation in the Linker Region of Reconstituted Positioned Nucleosomes by Tet1 Protein.

Tet (ten-eleven translocation) family proteins oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), and are suggested to be involved in the active DNA demethylation pathway. In this study, we reconstituted positioned mononucleosomes using CpG-methylated 382 bp DNA containing the Widom 601 sequence and recombinant histone octamer, and subjected the nucleosome to treatment with Tet1 protein. The sites of oxidized methylcytosine were identified by bisulfite sequencing. We found that, for the oxidation reaction, Tet1 protein prefers mCs located in the linker region of the nucleosome compared with those located in the core region.

Locating the uracil-5-yl radical formed upon photoirradiation of 5-bromouracil-substituted DNA.

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.

Advancing small-molecule-based chemical biology with next-generation sequencing technologies.

Next-generation-sequencing (NGS) technologies enable us to obtain extensive information by deciphering millions of individual DNA sequencing reactions simultaneously. The new DNA-sequencing strategies exceed their precursors in output by many orders of magnitude, resulting in a quantitative increase in valuable sequence information that could be harnessed for qualitative analysis. Sequencing on this scale has facilitated significant advances in diverse disciplines, ranging from the discovery, design, and evaluation of many small molecules and relevant biological mechanisms to maturation of personalized therapies. NGS technologies that have recently become affordable allow us to gain in-depth insight into small-molecule-triggered biological phenomena and empower researchers to develop advanced versions of small molecules. In this review we focus on the overlooked implications of NGS technologies in chemical biology, with a special emphasis on small-molecule development and screening.

Sequence-specific electron injection into DNA from an intermolecular electron donor.

Electron transfer in DNA has been intensively studied to elucidate its biological roles and for applications in bottom-up DNA nanotechnology. Recently, mechanisms of electron transfer to DNA have been investigated; however, most of the systems designed are intramolecular. Here, we synthesized pyrene-conjugated pyrrole-imidazole polyamides (PPIs) to achieve sequence-specific electron injection into DNA in an intermolecular fashion. Electron injection from PPIs into DNA was detected using 5-bromouracil as an electron acceptor. Twelve different 5-bromouracil-containing oligomers were synthesized to examine the electron-injection ability of PPI. Product analysis demonstrated that the electron transfer from PPIs was localized in a range of 8 bp from the binding site of the PPIs. These results demonstrate that PPIs can be a useful tool for sequence-specific electron injection.

Next-generation sequencing studies guide the design of pyrrole-imidazole polyamides with improved binding specificity by the addition of ß-alanine.

The identification of binding sites for small molecules in genomic DNA is important in various applications. Previously, we demonstrated rapid transcriptional activation by our small molecule SAHA-PIP. However, it was not clear whether the strong biological effects exerted by SAHA-PIP were attributable to its binding specificity. Here, we used high-throughput sequencing (Bind-n-seq) to determine the binding specificity of SAHA-PIPs. Sequence specificity bias was determined for SAHA-PIPs (3 and 4), and this showed enhanced 6 bp sequence-specific binding compared with hairpin PIPs (1 and 2). This finding allowed us to investigate the role of the ß-alanine that links SAHA to PIP, and led in turn to the design of ßß-PIPs (5 and 6), which showed enhanced binding specificity. Overall, we demonstrated the importance of ß-moieties for the binding specificity of PIPs and the use of cost-effective high-throughput screening of these small molecules for binding to the DNA minor groove.