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

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• G-quadruplexes (G4s) are four-stranded DNA structures with significant biological roles.
• Rap1-interacting factor 1 (Rif1) binds to Rif1-binding sequences (Rif1BS) and regulates DNA replication timing.
• Rif1BS contains multiple G-tracts, suggesting potential for complex DNA structures.

Purpose of the Study:

• To elucidate the precise molecular architecture of higher-order DNA structures formed at Rif1BS.
• To investigate the structural behavior of both DNA strands within Rif1BS upon heat denaturation.

Main Methods:

• Gel-shift DNA binding assays.
• DNA footprinting using various nuclease probes.
• Analysis of DNA structures under different conditions, including mutations and deazaguanine replacement.

Main Results:

• Both G-strand and C-strand of Rif1BS adopt specific higher-order structures upon heat denaturation.
• The G-strand forms a G4 structure with extended loops, while the C-strand forms a non-i-motif structure dependent on G4 formation.
• Multiple G-tracts allow for alternative structures, demonstrating the robustness of these DNA higher-order structures.

Conclusions:

• Higher-order DNA structures at Rif1BS involve both strands, with potential for G4 formation on both.
• The formation of structures on the C-strand is contingent upon G4 formation on the G-strand.
• These findings offer general insights into DNA structures formed at G4-forming sequences within duplex DNA.