Abstract
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.
Highlights
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
Rif1binding sequence (Rif1BS) contains at least two Rif1CS, with head-to-tail orientation in 75% of the cases, and the two Rif1CS are separated by a ϳ100-bp segment on average
We previously showed that the 3G-tracts in addition to Rif1CS are important for formation of G4 structures in the ssDNA and for in vivo chromatin binding of Rap1-interacting factor 1 (Rif1) [25]
Summary
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. G4 structures formed on ssDNA have been extensively studied, and diverse structures and topologies have been described at the atomic level [24] Those generated on duplex DNA have been poorly understood despite the highly prevalent presence of potential G4 structures on duplex genomes. The complementary strand of G-rich sequences capable of forming G4 has a capacity to generate i-motif, it has not been clear whether these two structures coexist on a duplex DNA. The nuclease sensitivity assays demonstrate the existence of unexpectedly long loop segments in the generated G4-like structure They indicate the generation of a higher-order structure on the other C-rich strand, which strictly depends on the G-tracts on the G strand and is not likely to be i-motif. We suggest that Rif1BS may generate G4 structures that may undergo dynamic transition from one structure to another through its potential to generate multiple G4-like structures on one or both strands
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