Human RTEL1 associates with Poldip3 to facilitate responses to replication stress and R-loop resolution

Abstract

RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/ function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.

RTEL1 Regulates G4/R-Loops to Avert Replication-Transcription Collisions

“…This is supported by our observation that overexpression of WT RNaseH1-GFP partially rescues the reduced fork extension rates, fork asymmetry, micronuclei, and 53BP1 and γH2AX foci in Rtel1 −/− ( Figures 3 A–3F) and Rtel1 PIP_box knockin ( Figures 3 I, 3J, and S4 A) cells. This implies that RTEL1 is important for the removal of R-loops in a PIP-box-dependent manner, which is consistent with a recent paper showing that Poldip3 facilitates RTEL1 recruitment to chromatin to remove R-loops ( Björkman et al., 2020 ). We previous established that increased replication fork stalling and/or collapse is the primary source of replication problems in Rtel1 −/− and Rtel1 PIP_box knockin cells ( Vannier et al., 2013 ).…”

“…However, the cause of fork stalling and/or collapse was unknown. Our findings and those of others ( Björkman et al., 2020 ; Takedachi et al., 2020 ; Wu et al., 2020 ) suggest that the replication defects in Rtel1 -deficient cells are primarily caused by TRCs resulting from inefficient removal of G4/R-loops. While the precise mechanism by which RTEL1 counteracts G4/R-loops remains to be defined, RTEL1 can unwind telomeric G4s in vitro ( Vannier et al., 2013 ), so it could use this activity to remove G4s within a G4/R-loop.…”

“…Several recent studies have also reported that depletion of RTEL1 leads to the accumulation of G4/R-loops ( Björkman et al., 2020 ; Wu et al., 2020 ). Notably, Wu et al.…”

Cell environment shapes TDP-43 function: implications in neuronal and muscle disease

“…Therefore, the results obtained in C2C12 suggest another possible mechanism on how TDP-43 may control gene expression in muscle in an indirect fashion and eventually participate in disease. Recently, loss of TDP-43 was associated with increased genomic instability and R-loop formation (Giannini et al, 2020;Wood et al, 2020) possibly through mechanisms involving Poldip3, which has been shown to play a role in maintaining genome stability and preventing R-loop accumulation at sites of active replication (Björkman et al, 2020).…”

The end protection problem—an unexpected twist in the tail

“…RTEL1 unwinds D-loops in vitro, while tethering of RTEL1 to telomeres via a TRF2-S365A mutant reduces t-loop levels in vivo, suggesting RTEL1 unwinds t-loops directly (Barber et al 2008;Vannier et al 2012). However, RTEL1 also unwinds telomeric G-quadruplex structures and possibly RNA-DNA hybrids (R-loops), although RTEL1 is recruited to these secondary structures through an interaction with PCNA, not with TRF2 (Vannier et al 2012;Björkman et al 2020;Wu et al 2020). Therefore, RTEL1 might also impact t-loops indirectly, by unwinding secondary structures that alter topological stress within the telomere.…”