Distinct roles of structure-specific endonucleases EEPD1 and Metnase in replication stress responses

Abstract

Accurate DNA replication and segregation are critical for maintaining genome integrity and suppressing cancer. Metnase and EEPD1 are DNA damage response (DDR) proteins frequently dysregulated in cancer and implicated in cancer etiology and tumor response to genotoxic chemo- and radiotherapy. Here, we examine the DDR in human cell lines with CRISPR/Cas9 knockout of Metnase or EEPD1. The knockout cell lines exhibit slightly slower growth rates, significant hypersensitivity to replication stress, increased genome instability and distinct alterations in DDR signaling. Metnase and EEPD1 are structure-specific nucleases. EEPD1 is recruited to and cleaves stalled forks to initiate fork restart by homologous recombination. Here, we demonstrate that Metnase is also recruited to stalled forks where it appears to dimethylate histone H3 lysine 36 (H3K36me2), raising the possibility that H3K36me2 promotes DDR factor recruitment or limits nucleosome eviction to protect forks from nucleolytic attack. We show that stalled forks are cleaved normally in the absence of Metnase, an important and novel result because a prior study indicated that Metnase nuclease is important for timely fork restart. A double knockout was as sensitive to etoposide as either single knockout, suggesting a degree of epistasis between Metnase and EEPD1. We propose that EEPD1 initiates fork restart by cleaving stalled forks, and that Metnase may promote fork restart by processing homologous recombination intermediates and/or inducing H3K36me2 to recruit DDR factors. By accelerating fork restart, Metnase and EEPD1 reduce the chance that stalled replication forks will adopt toxic or genome-destabilizing structures, preventing genome instability and cancer. Metnase and EEPD1 are overexpressed in some cancers and thus may also promote resistance to genotoxic therapeutics.

RAD52: Paradigm of Synthetic Lethality and New Developments

“…(C) RAD52 also recognizes and repairs stalled replication forks via break-induced replication (BIR). The structure is cleaved by the endonuclease complex MUS81 and processed by EEPD1 ( Kim et al, 2017 ; Sharma et al, 2020 ). Bound to the one-ended DNA break, RAD52 invades the dsDNA to form a D-loop.…”

The Safe Path at the Fork: Ensuring Replication-Associated DNA Double-Strand Breaks are Repaired by Homologous Recombination

“…Template switching uses sister chromatids to bypass blocking lesions and is generally error-free ( Figure 2A ), but poses risks of genome rearrangement from replisome switching to non-sister templates ( Lehmann et al, 2020 ). Two additional restart pathways are fork reversal to a Holliday junction-like structure followed by fork restoration, and fork cleavage by structure-specific nucleases including the 3′ nuclease MUS81 (with EME2) ( Pepe and West, 2014 ) and the 5’ nuclease EEPD1 ( Wu et al, 2015 ; Sharma et al, 2020 ) ( Figure 2B ). Metnase is a structure-specific nuclease that promotes fork restart, but Metnase does not cleave stalled forks and instead may process flaps that arise later ( Sharma et al, 2020 ).…”

“…Two additional restart pathways are fork reversal to a Holliday junction-like structure followed by fork restoration, and fork cleavage by structure-specific nucleases including the 3′ nuclease MUS81 (with EME2) ( Pepe and West, 2014 ) and the 5’ nuclease EEPD1 ( Wu et al, 2015 ; Sharma et al, 2020 ) ( Figure 2B ). Metnase is a structure-specific nuclease that promotes fork restart, but Metnase does not cleave stalled forks and instead may process flaps that arise later ( Sharma et al, 2020 ). SLX1-SLX4 is another structure-specific nuclease that resolves branched replication intermediates, and although it cleaves many types of branched structures including replication fork structures in vitro , direct evidence that it cleaves stalled forks in vivo is lacking ( Falquet and Rass, 2019 ; Xu et al, 2021 ).…”

Structure, Activity, and Function of SETMAR protein Lysine Methyltransferase

“…SETMAR has also been proposed to act in DNA replication and cell cycle regulation (Figure 4A). Knockdown, knockout, or overexpression of SETMAR has more or less of an effect on cell cycle depending on the cell type, with generally a higher SETMAR expression correlating with an increased growth rate [16,19,21,44,61]. While phosphorylation of SETMAR on the Ser508 residue by Chk1 is associated with DNA damage repair via NHEJ, the unphosphorylated form of SETMAR is involved in the response to replication stress [47].…”

“…The endonuclease activity of SETMAR is thought to be involved in the cleavage of branched DNA structures resulting from stress replication forks, producing DSBs that can be resolved by DNA damage repair pathways and thus allowing the restart of replication forks [16,40,62,63]. A recent work has proposed that SETMAR role in the response to replication stress was mediated by the dimethylation of H3K36 at stalled replication forks, facilitating the recruitment of DNA repair factors, rather than a cleavage of stalled forks via SETMAR endonuclease activity [61]. It has been recently suggested that SETMAR could bind to 12 bp motifs that are enriched in the regions of replication origins but more work is required to determine whether SETMAR binding is occurring simultaneously to DNA replication and whether SETMAR binding plays a role in DNA replication [34].…”