Project description:The molecular mechanism for ssRNA+dsDNA triple helix structure formation on chromatin are unclear. We analyzed the triplex-nucleosomes complex fomration in vitro and in vivo, and show that triplexes are stabilized by the nucleosomes. We developed a method to monitor nucleosome bound RNA triplexes in vivo, revealing that RNA binding maintained the nucleosomes on an accessible structure supporting a gene activating role of nucleosome-triplex complexes in cells.
Project description:We took advantage of ssRNA-seq technology to deeply sequence mRNAs of the model plant species Oryza sativa ssp.japonica cv Nipponbare with clear transcriptional orientations for assessing rice cis-NATs at the best possible resolution. We also deeply sequenced rice small RNAs from the same tissues as that for preparing mRNAs to investigate rice cis-NAT pairs that potentially give rise to endogenous short interfering RNAs from their overlapping regions under normal and stress conditions.
Project description:In a first step of DNA double-strand break (DSB) repair by homologous recombination, DNA ends are resected such that single-stranded DNA (ssDNA) overhangs are generated. ssDNA is specifically bound by RPA and other factors, which constitutes a ssDNA-compartment on damaged chromatin. The molecular organization of this ssDNA- as well as the adjacent dsDNA-compartment is crucial during DSB signaling and repair. However, data regarding the association of the most basic chromatin components – the nucleosomes – have been discrepant. Here, we use site-specific induction of DSBs and chromatin-immunoprecipitation followed by strand-specific sequencing to analyse in vivo binding of key DSB repair and signalling proteins to either the ssDNA- or dsDNA-compartment. In case of nucleosomes, we show that recently proposed ssDNA-nucleosomes are not a major, persistent species, but that nucleosomes eviction and DNA end resection are intrinsically coupled. These results support a model of separated dsDNA-nucleosome- and ssDNA-RPA-compartments during DSB signaling and repair.
Project description:Transfection of dsDNA into many mammalian cell types indues the production of type I interferons and interferon-stimulated genes. We performed an siRNA screen to identify genes involved in this innate immune response, and identified Abcf1. We used microarrays to determine which genes are regulated by ABCF1 following dsDNA stimulation. We treated p53-/- mouse embryonic fibroblasts (MEFs) with siRNAs targeting Abcf1 or Irf3 (positive control) or a negative control siRNA (siCtrl), and then stimulated the cells with 45-base pair dsDNA for 6 hrs or left the cells unstimulated. We then lysed the cells and hybridized the RNA to Affymetrix microarrays.
Project description:The replication initiation proteins interact dsDNA located at replication origin and ssDNA of DNA unwinding element (DUE), formed as a result of the destabilization of the double-stranded helix of AT-rich origin region. It is a critical step in the DNA replication initiation; however, the structure of nucleoprotein complex involving initiator protein, dsDNA and/or ssDNA is still elusive and different models are proposed. In this work, based on crosslinking combined with mass spectrometry (MS), structural and bioinformatic analysis, we defined amino acid residues in plasmid Rep proteins, TrfA and RepE, that are essential for interaction with ssDNA. The study of Rep mutant proteins containing single amino acid substitutions affecting DNA interaction reveals the importance of Rep-ssDNA complexes formation for a dsDNA melting at DUE. Furthermore, the crystal structures obtained for complex of RepE protein with DUE ssDNA, and, RepE complexed with both DUE ssDNA and dsDNA containing RepE specific binding site (iteron) revealed that the plasmid initiator can not only bind iterons and ssDNA DUE separately but also can form a tripartite nucleoprotein complex bringing together specific sequences of replication origin. The presented data strongly supports the loop-back model in which a replication initiator molecule interacts with dsDNA and ssDNA.