title: Chemical and biological approaches to improve the efficiency of homologous recombination in human cells mediated by artificial restriction DNA cutter.
abstract: A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3'-overhang termini provided by 1.7-fold higher efficiency than 5'-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.
authors.affiliation: Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Katada
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Harumoto
authors.forename: Narumi
authors.initials: N
authors.lastname: Shigi
authors.forename: Makoto
authors.initials: M
authors.lastname: Komiyama
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics; D002453:Cell Cycle; D002460:Cell Line; D002563:Cerium / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D004492:Edetic Acid / Q000737:chemistry*; D059765:Homologous Recombination; D006801:Humans; D000072200:Ku Autoantigen; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D034622:RNA Interference; D012689:Sequence Homology, Nucleic Acid
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D020135:Peptide Nucleic Acids; D002563:Cerium; D004247:DNA; D004492:Edetic Acid; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen