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

Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila, 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 a physical 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 an 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 direct regulator of replication fork progression suggesting developmental regulation of Rif1 activity. Copy number profiles from third instar salivary gland DNA in WT, SuUR, SuUR(deltaSNF) and Rif1 mutants. Once replicate is included for WT, SuUR and SuUR(deltaSNF). Two replicates are included for the Rif1 mutant. Additionally, copy number profile of stage 13 egg chamber DNA. One replicate for WT and SuUR mutant. Two replicates for Rif1 mutant.