Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila

Alexander Munden,Zhan Rong,Simon Mallal,Amanda Sun,Jared T Nordman,Rama Gangula

doi:10.7554/elife.39140

Abstract

Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR's SNF2 domain, we identified an interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in a partially SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression.

Highlights

Accurate duplication of a cell’s genetic information is essential to maintain genome stability
To study the function of SUUR’s SNF2 domain, we generated a mutant in which the SNF2 domain was deleted and the resulting mutant protein was expressed under the control of the endogenous Suppressor of Underreplication (SuUR) promoter
SUUR serves as a scaffold to recruit a Rap1 interacting factor 1 (Rif1)/Phosphatase 1 (PP1) complex to replication forks where Rif1/PP1 inhibits replication fork progression through dephosphorylation of a component of the replisome

Summary

Introduction

Accurate duplication of a cell’s genetic information is essential to maintain genome stability. Rif has been show to interact with Lamin and is thought to tether specific regions of the genome to the nuclear periphery (Foti et al, 2016) How this activity is related to Rif1’s ability to inactivate helicases together with PP1 in controlling the timing program remains obscure. Function results in increased replication fork progression, overexpression of SUUR drastically inhibits fork progression without affecting origin firing (Nordman et al, 2014; Sher et al, 2012) These findings, together with previous work showing that loss of SUUR function has no influence on ORC binding (Sher et al, 2012) and that SUUR associates with euchromatin in an S phase-dependent manner (Kolesnikova et al, 2013), further supports SUUR as a direct inhibitor of replication fork progression within specific regions of the genome. Our findings provide mechanistic insight into the process of underreplication and define a new function for Rif in replication control

Results

Discussion

Materials and methods

Funding Funder National Institutes of Health