TonEBP Regulates PCNA Polyubiquitination in Response to DNA Damage through Interaction with SHPRH and USP1

Abstract

Summary Polyubiquitination of proliferating cell nuclear antigen (PCNA) regulates the error-free template-switching mechanism for the bypass of DNA lesions during DNA replication. PCNA polyubiquitination is critical for the maintenance of genomic integrity; however, the underlying mechanism is poorly understood. Here, we demonstrate that tonicity-responsive enhancer-binding protein (TonEBP) regulates PCNA polyubiquitination in response to DNA damage. TonEBP was recruited to DNA damage sites with bulky adducts and sequentially recruited E3 ubiquitin ligase SHPRH, followed by deubiquitinase USP1, to DNA damage sites, in correlation with the dynamics of PCNA polyubiquitination. Similarly, TonEBP was found to be required for replication fork protection in response to DNA damage. The Rel-homology domain of TonEBP, which encircles DNA, was essential for the interaction with SHPRH and USP1, PCNA polyubiquitination, and cell survival after DNA damage. The present findings suggest that TonEBP is an upstream regulator of PCNA polyubiquitination and of the DNA damage bypass pathway.

Tonicity-responsive enhancer-binding protein promotes stemness of liver cancer and cisplatin resistance

“…To induce HCC, we administrated a single intraperitoneal injection of 25 mg/kg diethylnitrosamine (DEN) (N0756; Sigma) to 2-week-old C57BL/6 mice and euthanised them at 9 months of age [25] .…”

“…Anti-ERCC1 antibodies (CST); anti-XPF antibodies (Abcam); anti-Myc antibodies (CST). ChIP was performed as previous studies [25] . In brief, cells were crosslinked with 1% formaldehyde followed by addition of 125 mM glycine.…”

“…We analyzed the candidate of TonEBP-interacting protein from the interactome data of previous report [25] . To isolate TonEBP-interacting proteins, HEK293 cells were transfected with the pcDNA5/FRT empty vector or pcDNA5/FRT-TonEBP-Flag plasmid.…”

“…TonEBP expression is stimulated by inflammation leading to elevated expression of pro-inflammatory genes and chronic inflammatory diseases [ [20] , [21] , [22] , [23] , [24] ]. In addition, TonEBP is recruited to the sites of DNA damage as an upstream regulator of DDR [25] . The same study also revealed that TonEBP interactome includes 250 proteins associated with DDR suggesting that TonEBP has more functions related to DDR.…”

Ubiquitin and Ubiquitin-Like Proteins Are Essential Regulators of DNA Damage Bypass

“…While it remains unclear how the switch from PCNA mono- to polyubiquitination is regulated, it is likely to involve the specific recruitment and activation of the Rad5 orthologues. For instance, in response to MMS-induced DNA damage, PCNA polyubiquitination seems to be dependent on SHPRH recruitment by TonEBP (tonicity-responsive enhancer-binding protein), a protein which itself binds to and encircles DNA following alkylation damage [ 54 ]. Recruitment of the Rad5 orthologues is, however, unlikely to be sufficient on its own for driving the switch to PCNA polyubiquitination, especially given findings that both of these proteins can also function in TLS to promote the binding of Y-family polymerases to monoubiquitinated PCNA.…”