A Novel Checkpoint and RPA Inhibitory Pathway Regulated by Rif1

Abstract

Cells accumulate single-stranded DNA (ssDNA) when telomere capping, DNA replication, or DNA repair is impeded. This accumulation leads to cell cycle arrest through activating the DNA–damage checkpoints involved in cancer protection. Hence, ssDNA accumulation could be an anti-cancer mechanism. However, ssDNA has to accumulate above a certain threshold to activate checkpoints. What determines this checkpoint-activation threshold is an important, yet unanswered question. Here we identify Rif1 (Rap1-Interacting Factor 1) as a threshold-setter. Following telomere uncapping, we show that budding yeast Rif1 has unprecedented effects for a protein, inhibiting the recruitment of checkpoint proteins and RPA (Replication Protein A) to damaged chromosome regions, without significantly affecting the accumulation of ssDNA at those regions. Using chromatin immuno-precipitation, we provide evidence that Rif1 acts as a molecular “band-aid” for ssDNA lesions, associating with DNA damage independently of Rap1. In consequence, small or incipient lesions are protected from RPA and checkpoint proteins. When longer stretches of ssDNA are generated, they extend beyond the junction-proximal Rif1-protected regions. In consequence, the damage is detected and checkpoint signals are fired, resulting in cell cycle arrest. However, increased Rif1 expression raises the checkpoint-activation threshold to the point it simulates a checkpoint knockout and can also terminate a checkpoint arrest, despite persistent telomere deficiency. Our work has important implications for understanding the checkpoint and RPA–dependent DNA–damage responses in eukaryotic cells.

Author Summary

Here we identified arguably the first anti-checkpoint protein in Rif1. The term anti-checkpoint was proposed by Ted Weinert, one of the parents of the checkpoint concept, to describe a factor that stops checkpoint proteins from responding to DNA damage by other means than repair, reduced amounts of ssDNA, or adaptation [1]. No such factor has been clearly identified; potential candidates (for example, shelterin or CST complexes at chromosome ends) may reduce the amount of damage, therefore exerting an indirect “anti-checkpoint” function. Interestingly, mammalian Rif1 was once thought to be a checkpoint protein [2]. Our study indicates that Rif1 out-competes checkpoint proteins for their substrate and sets a threshold for checkpoint activation in budding yeast. Rif1 can tune down the checkpoint responses, thus permitting cells to proliferate with DNA damage, a pre-requisite for chromosomal instability, the hallmark of cancer cells. Rif1 is an important link in understanding how eukaryotic cells balance the need to proliferate with the need to preserve their genetic heritage. Finding an anti-checkpoint is not of pure theoretical interest. In the future, Rif1 inhibitors could limit proliferation of chromosomally unstable cells. Conversely, Rif1 enhancers could tune down the over-blown checkpoint responses that lead to massive cell death following different insults.