Removal of RTF2 from Stalled Replisomes Promotes Maintenance of Genome Integrity

Abstract

The protection and efficient restart of stalled replication forks is critical for the maintenance of genome integrity. Here, we identify a regulatory pathway that promotes stalled forks recovery from replication stress. We show that the mammalian replisome component C20orf43/RTF2 (homologous to S. pombe Rtf2) must be removed for fork restart to be optimal. We further show that the proteasomal shuttle proteins DDI1 and DDI2 are required for RTF2 removal from stalled forks. Persistence of RTF2 at stalled forks results in fork restart defects, hyperactivation of the DNA damage signal, accumulation of single-stranded DNA (ssDNA), sensitivity to replication drugs, and chromosome instability. These results establish that RTF2 removal is a key determinant for the ability of cells to manage replication stress and maintain genome integrity.

The yeast proteases Ddi1 and Wss1 are both involved in the DNA replication stress response

“…Many publications describing yeast Ddi1 have focused on its role as a proteasomal shuttling protein, bringing polyubiquitinated substrates to the 26S proteasome (Clarke et al 2001;Finley 2009;Ivantsiv et al 2006;Kaplun et al 2005;Voloshin et al 2012). A shuttling mechanism has also been proposed for human Ddi1-like proteins in the degradation of RTF2 and subsequent restart of replication forks (Kottemann et al 2018). In contrast, our data do not support the proteasome shuttling mechanism, as deletion of UBL, UBA or both did not impair the ability of the protein to rescue hydroxyurea sensitivity.…”

“…As part of a genome-wide synthetic lethality screen in yeast, Ddi1 exhibited a strong negative genetic interaction with Wss1, another replication stress-countering protease (Costanzo et al 2016). Another study identified human proteins DDI1 and DDI2, mammalian orthologues of Ddi1 that are involved in degradation of the replication termination factor RTF2, facilitating restart of stalled replication forks (Kottemann et al 2018).…”

“…Other proteases recently found to be involved in the replication stress response include the Ddi1-like proteins. Mammalian DDI1 and DDI2 proteins associate with nascent replication forks and are involved in restarting stalled forks by facilitating removal of the replication termination factor RTF2 (Kottemann et al 2018). DDI1-and DDI2-depleted cells fail to remove RTF2 from stalled forks, leading to accumulation of single-stranded DNA, sensitivity to replication drugs, and chromosome instability.…”

Crystal structure of the retroviral protease‐like domain of a protozoal DNA damage‐inducible 1 protein

“…The RMSD values of the flap of one subunit (subunit A) increased over the course of the simulation and stabilized at about 4. 5 A, whereas that of the flap of the other subunit (subunit B) showed a similar increase during the initial duration but eventually decreased and stabilized at about 3 A near the end of the 300 ns run (Fig. 6A).…”

“…But when one subunit of each Ddi1-RVP dimer is superimposed, the second subunits deviate in position with RMSD values of 1.0-1. 5 A due to slightly differing angles (2.3-5.4°) between the two subunits in each dimer, suggesting a small relative movement of the subunits (Fig. 9).…”