Rif1 maintains telomeres and mediates DNA repair with its N-terminal alpha-helical repeat

Rif1 plays a central role in genome maintenance ranging from telomere length regulation in budding yeast, to DNA double-strand break (DSB) repair pathway choice in mammals, and replication timing regulation in both organisms. In yeast, Rif1 controls telomere length by inhibiting telomerase and checkpoint signaling at chromosome ends. In mammals, RIF1 has emerged as a critical regulator of genome stability, mediating DSB repair pathway choice by attenuating DNA end resection. RIF1 thereby inhibits homologous recombination and promotes non-homologous end joining (NHEJ). In contrast, the involvement of Rif1 at DSBs in budding yeast remains controversial. The N-terminal domain (NTD) is the most conserved and largest folded domain within Rif1 and is predicted to form an alpha-helical repeat. Although Rif1 has been studied extensively, our molecular understanding remains limited, and it is puzzling how an alpha-helical repeat may fulfill such a wide range of functions. In my Ph.D. research, I examined the Rif1-NTD in budding yeast and human using structural, biochemical, and biophysical approaches to shed light on their structure and function at the molecular level and to characterize their interactions with binding partners.
In the first part of my doctoral studies (Chapter 2), we determined the crystal structure of the conserved 125 kDa budding yeast Rif1-NTD. Our structure revealed an extended architecture spanning 238 Å in length that consists of an irregular alpha-helical repeat. Using biochemical approaches, we identified Rif1-NTD as a high-affinity DNA-binding protein and determined the co-crystal structure of Rif1-NTD in complex with dsDNA. The co-crystal structure shows that a 16 bp DNA footprint is encased by the Rif1-NTD concave surface and that Rif1-NTD forms a figure-8-shaped head-to-tail dimer in the presence of DNA. This arrangement was confirmed by negative stain electron microscopy. Site-directed mutagenesis at the Rif1 DNA-binding interface reduced the binding affinity in vitro. In vivo, tight DNA association proved essential for checkpoint control and telomere length regulation. Surprisingly, Rif1-NTD also bound to uncapped telomeres and DSBs, thereby inhibiting end resection and promoting NHEJ in budding yeast. Decreasing the DNA binding affinity caused a loss of function phenotype for the inhibition of resection and DSB repair pathway choice. Thus, the direct association of the Rif1-NTD with DNA is required for DSB repair pathway choice in budding yeast. This finding demonstrates that the role of Rif1 in DSB repair pathway choice is conserved from yeast to human and provides first insights into the function of the Rif1-NTD at the molecular level.
In the second part of my doctoral studies (Chapter 3), we turned our attention to the human RIF1-NTD. While localization to DSBs is mediated by the direct interaction with DNA in budding yeast, human RIF1 recruitment to DSBs is strictly dependent on phosphorylated 53BP1. When recruitment of RIF1 is hampered, DSBs are not protected from resection and NHEJ cannot occur. However, it remained elusive whether RIF1 and 53BP1 interact directly or whether they require a mediator protein that may be able to distinguish between the different 53BP1 phospho-states. Unexpectedly, we found that the 53BP1-Rif1 interaction is direct, that it is mediated by the RIF1-NTD, and that complex formation is strictly dependent on a phosphorylated 53BP1 motif. This finding is remarkable because RIF1-NTD is an alpha-helical repeat and does not contain a domain known for phospho-specific interactions. Our biophysical data dissect RIF1 recruitment to 53BP1 at the molecular level and will inform future structural and functional characterization of the 53BP1-RIF1 complex, which is required to block DNA end resection and trigger NHEJ.
In summary, we have shown that the Rif1-NTD forms an elongated alpha-helical repeat and that Rif1-NTD maintains telomeres and mediates DSB repair pathway choice in budding yeast. Rif1-NTD accomplishes these functions through its direct interaction with DNA thereby blocking DNA end resection. In human, RIF1-NTD also blocks DNA end resection, and we have shown that RIF1-NTD directly interacts with 53BP1 in a phosphorylation dependent manner. Although Rif1 uses different mechanisms across species, Rif1 blocks DNA end resection in budding yeast and human – a conserved function mediated by its N-terminal alpha-helical repeat.