Rif1-Dependent Control of Replication Timing

Abstract

Successful duplication of the genome requires the accurate replication of billions of base pairs of DNA within a relatively short time frame. Failure to accurately replicate the genome results in genomic instability and a host of diseases. To faithfully and rapidly replicate the genome, DNA replication must be tightly regulated and coordinated with many other nuclear processes. These regulations, however, must also be flexible as replication kinetics can change through development and differentiation. Exactly how DNA replication is regulated and how this regulation changes through development is an active field of research. One aspect of genome duplication where much remains to be discovered is replication timing (RT), which dictates when each segment of the genome is replicated during S phase. All organisms display some level of RT, yet the precise mechanisms that govern RT remain are not fully understood. The study of Rif1, a protein that actively regulates RT from yeast to humans, provides a key to unlock the underlying molecular mechanisms controlling RT. The paradigm for Rif1 function is to delay helicase activation within certain regions of the genome, causing these regions to replicate late in S phase. Many questions, however, remain about the intricacies of Rif1 function. Here, we review the current models for the activity of Rif1 with the goal of trying to understand how Rif1 functions to establish the RT program.

Keywords: DNA replication; Rif1; genome stability; helicase; nuclear organization; replication timing.

Figure 1

The functions of replication timing. (A) The replication timing program maintains a supply–demand equilibrium of limiting replication factors, which could be histones, replisome proteins or dNTPs. In a de-regulated replication timing program, an excess of origins may become activated, resulting in the pool of limiting replication factors to be depleted and DNA damage to occur. (B) Late-replicating regions have a higher mutation rate than early-replicating regions. (C) The replication timing program maintains the epigenetic landscape. In the wild-type example (blue box), replication timing preserves the epigenetic landscape. This is achieved by allowing euchromatic and heterochromatic histone readers and writers to be properly recruited to replication forks within early and late-replicating regions, respectively, during S phase. The pink boxes indicate de-regulated replication timing. In this example, a region switches from early to late replicating (E to L Switch) or late to early replicating (L to E switch). Consequently, the wrong histone readers and writers are targeted to those regions. This results in the epigenetic landscape changing upon replication timing disruption.

Figure 2

The functions of Rif1. (A) In G1 phase, Rif1 may reverse the CDK-mediated phosphorylation of ORC to promote ORC-dependent loading of MCMs. (B) In S phase, Rif1 opposes DDK-mediated MCM phosphorylation to inhibit helicase activation and origin activation in late-replicating genomic regions. (C) A representative view of Rif1 genomic binding on a chromosomal arm. Green boxes denote origins of replication, where Rif1 genomic binding has a sharp, well-defined peaks. Black boxes indicate regions of heterochromatin, where Rif1 binds to broad domains with a lower signal intensity compared to origins. The broad Rif1 binding domains also have overlap with lamin-associated domains, indicated by blue boxes.

Figure 3

Rif1 mediates nuclear organization. In cells that are wild type for Rif1 (blue box), early and late replicating domains are separated from each other (green and red DNA respectively). The late replicating domains, which are coated with Rif1, have limited physical interactions. In cells that are mutant for Rif1, there is an increase in physical interactions between genomic domains that depend on Rif1 to maintain their replication timing (Adapted from Foti et al., 2016 [65]).