Rif1 binds to G quadruplexes and suppresses replication over long distances

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Rif1 protein plays a crucial role in regulating DNA replication timing and repairing DNA double-strand breaks.
• Understanding the precise mechanisms of Rif1's function is essential for comprehending genome stability and cell cycle control.

Purpose of the Study:

• To identify and characterize the DNA binding sites of Rif1 in fission yeast.
• To elucidate the molecular basis of Rif1 recognition and its impact on replication origin activity.

Main Methods:

• Chromatin immunoprecipitation sequencing (ChIP-seq) was employed to map Rif1 binding sites across fission yeast chromosomes.
• In vitro biochemical assays were used to analyze the structural properties of Rif1 binding sites and Rif1-DNA interactions.

Main Results:

• Thirty-five high-affinity Rif1 binding sites were identified, predominantly located near dormant replication origins.
• A conserved DNA motif (CNWWGTGGGGG) was found at these sites, and mutations abolished Rif1 binding and activated late-firing origins.
• Rif1 binding sites exhibit G quadruplex-like structures in vitro, which are preferentially bound by purified Rif1.

Conclusions:

• Rif1 recognizes and binds to G quadruplex-like DNA structures at specific intergenic regions.
• This interaction establishes local chromatin structures that suppress the firing of nearby replication origins, influencing replication timing.