Probing the Potential Role of Non-B DNA Structures at Yeast Meiosis-Specific DNA Double-Strand Breaks

• Published in: Biophysical journal Vol. 112; no. 10; pp. 2056 - 2074
• Main Authors: Kshirsagar, Rucha, Khan, Krishnendu, Joshi, Mamata V., Hosur, Ramakrishna V., Muniyappa, K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23-05-2017; Biophysical Society; The Biophysical Society
• Subjects: Abundance; Affinity; Alignment; Amino acid sequence; Baking yeast; Cell division; Chromatids; Circular Dichroism; Deoxyribonucleic acid; DNA; DNA Breaks, Double-Stranded; DNA damage; DNA polymerase; DNA, Single-Stranded; DNA-Binding Proteins - metabolism; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - metabolism; Double-strand break repair; Escherichia coli; Fluorescence; G-Quadruplexes; GC Rich Sequence; Gene expression; Genomes; Green fluorescent protein; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Guanine; In vitro methods and tests; Meiosis; Meiosis - genetics; Meiosis - physiology; Microscopy, Confocal; Mutation; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acids and Genome Biophysics; Proteins; Recombination; RNA, Messenger - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - metabolism; Segments; Sister chromatids; Substrate specificity; Synaptonemal complex; Yeast; Yeasts
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2017.04.028

A plethora of evidence suggests that different types of DNA quadruplexes are widely present in the genome of all organisms. The existence of a growing number of proteins that selectively bind and/or process these structures underscores their biological relevance. Moreover, G-quadruplex DNA has been implicated in the alignment of four sister chromatids by forming parallel guanine quadruplexes during meiosis; however, the underlying mechanism is not well defined. Here we show that a G/C-rich motif associated with a meiosis-specific DNA double-strand break (DSB) in Saccharomyces cerevisiae folds into G-quadruplex, and the C-rich sequence complementary to the G-rich sequence forms an i-motif. The presence of G-quadruplex or i-motif structures upstream of the green fluorescent protein-coding sequence markedly reduces the levels of gfp mRNA expression in S. cerevisiae cells, with a concomitant decrease in green fluorescent protein abundance, and blocks primer extension by DNA polymerase, thereby demonstrating the functional significance of these structures. Surprisingly, although S. cerevisiae Hop1, a component of synaptonemal complex axial/lateral elements, exhibits strong affinity to G-quadruplex DNA, it displays a much weaker affinity for the i-motif structure. However, the Hop1 C-terminal but not the N-terminal domain possesses strong i-motif binding activity, implying that the C-terminal domain has a distinct substrate specificity. Additionally, we found that Hop1 promotes intermolecular pairing between G/C-rich DNA segments associated with a meiosis-specific DSB site. Our results support the idea that the G/C-rich motifs associated with meiosis-specific DSBs fold into intramolecular G-quadruplex and i-motif structures, both in vitro and in vivo, thus revealing an important link between non-B form DNA structures and Hop1 in meiotic chromosome synapsis and recombination.