title: Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay.
abstract: 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.
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-Ku, Kyoto 606-8502, Japan.
authors.forename: Abhijit
authors.initials: A
authors.lastname: Saha
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-Ku, Kyoto 606-8502, Japan.
authors.forename: Seiichiro
authors.initials: S
authors.lastname: Kizaki
authors.affiliation: Department of Molecular Cell Biology, Sungkyunkwan University, School of Medicine, Suwon 440-746, Korea.
authors.forename: Debojyoti
authors.initials: D
authors.lastname: De
authors.affiliation: Institute for Integrated Cell-Materials Sciences (iCeMS) Kyoto University, Yoshida-ushinomiyacho, Sakyo-Ku, Kyoto 606-8501, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Endo
authors.affiliation: Department of Molecular Cell Biology, Sungkyunkwan University, School of Medicine, Suwon 440-746, Korea kyeongkyu@skku.edu.
authors.forename: Kyeong Kyu
authors.initials: KK
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-Ku, Kyoto 606-8502, Japan Institute for Integrated Cell-Materials Sciences (iCeMS) Kyoto University, Yoshida-ushinomiyacho, Sakyo-Ku, Kyoto 606-8501, Japan hs@kuchem.kyoto-u.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sugiyama
mesh_terms: D001483:Base Sequence; D001976:Bromouracil / Q000031:analogs & derivatives; D004247:DNA / Q000378:metabolism; D053837:DNA Cleavage / Q000528:radiation effects; D004583:Electrons; D004742:Enhancer Elements, Genetic; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D008027:Light; D000071841:PAX6 Transcription Factor / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000528:radiation effects; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D015203:Reproducibility of Results; D055748:SOXB1 Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000378:metabolism; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D055748:SOXB1 Transcription Factors; D019271:Hypoxanthine; D001976:Bromouracil; D014364:Tryptophan; D004247:DNA; C006824:5-bromouridine; D014529:Uridine