Open AccessChemical and biological approaches to improve the efficiency of homologous recombination in human cells mediated by artificial restriction DNA cutter
Author(s) -
Hitoshi Katada,
Toshimasa Harumoto,
Narumi Shigi,
Makoto Komiyama
Publication year - 2012
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gks185
Subject(s) - biology , dna , homologous recombination , non homologous end joining , nucleic acid , nuclease , recombination , microbiology and biotechnology , biochemistry , biophysics , computational biology , gene
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.