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Molecular architecture of G-quadruplex structures generated on duplex Rif1-binding sequences.

J Biol Chem. 2018 Nov 02;293(44):17033-17049. Epub 2018 Sep 14
Hisao Masai 1 , Naoko Kakusho 2 , Rino Fukatsu 2 , Yue Ma 3 , Keisuke Iida 4 , Yutaka Kanoh 2 , Kazuo Nagasawa 3
Hisao Masai 1 , Naoko Kakusho 2 , Rino Fukatsu 2 , Yue Ma 3 , Keisuke Iida 4 , Yutaka Kanoh 2 , Kazuo Nagasawa 3
+ et al

[No authors listed]

Author information
  • 1 From the Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan, masai-hs@igakuken.or.jp.
  • 2 From the Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan.
  • 3 the Department of Biotechnology and Life Science, Faculty of Technology, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan, and.
  • 4 the Molecular Chirality Research Center, Synthetic Organic Chemistry, Department of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, Japan.

摘要


G-quadruplexes (G4s) are four-stranded DNA structures comprising stacks of four guanines, are prevalent in genomes, and have diverse biological functions in various chromosomal structures. A conserved protein, Rap1-interacting factor 1 (Rif1) from fission yeast (Schizosaccharomyces pombe), binds to Rif1-binding sequence (Rif1BS) and regulates DNA replication timing. Rif1BS is characterized by the presence of multiple G-tracts, often on both strands, and their unusual spacing. Although previous studies have suggested generation of G4-like structures on duplex Rif1BS, its precise molecular architecture remains unknown. Using gel-shift DNA binding assays and DNA footprinting with various nuclease probes, we show here that both of the Rif1BS strands adopt specific higher-order structures upon heat denaturation. We observed that the structure generated on the G-strand is consistent with a G4 having unusually long loop segments and that the structure on the complementary C-strand does not have an intercalated motif (i-motif). Instead, we found that the formation of the C-strand structure depends on the G4 formation on the G-strand. Thus, the higher-order structure generated at Rif1BS involved both DNA strands, and in some cases, G4s may form on both of these strands. The presence of multiple G-tracts permitted the formation of alternative structures when some G-tracts were mutated or disrupted by deazaguanine replacement, indicating the robust nature of DNA higher-order structures generated at Rif1BS. Our results provide general insights into DNA structures generated at G4-forming sequences on duplex DNA.

KEYWORDS: DNA replication, DNase I, G-quadruplex, Rif1, S1 nuclease, chromosomes, non-B DNA, nuclease footprinting, nucleic acid, nucleic acid structure