Metnase and EEPD1: DNA Repair Functions and Potential Targets in Cancer Therapy

Abstract

Cells respond to DNA damage by activating signaling and DNA repair systems, described as the DNA damage response (DDR). Clarifying DDR pathways and their dysregulation in cancer are important for understanding cancer etiology, how cancer cells exploit the DDR to survive endogenous and treatment-related stress, and to identify DDR targets as therapeutic targets. Cancer is often treated with genotoxic chemicals and/or ionizing radiation. These agents are cytotoxic because they induce DNA double-strand breaks (DSBs) directly, or indirectly by inducing replication stress which causes replication fork collapse to DSBs. EEPD1 and Metnase are structure-specific nucleases, and Metnase is also a protein methyl transferase that methylates histone H3 and itself. EEPD1 and Metnase promote repair of frank, two-ended DSBs, and both promote the timely and accurate restart of replication forks that have collapsed to single-ended DSBs. In addition to its roles in HR, Metnase also promotes DSB repair by classical non-homologous recombination, and chromosome decatenation mediated by TopoIIα. Although mutations in Metnase and EEPD1 are not common in cancer, both proteins are frequently overexpressed, which may help tumor cells manage oncogenic stress or confer resistance to therapeutics. Here we focus on Metnase and EEPD1 DNA repair pathways, and discuss opportunities for targeting these pathways to enhance cancer therapy.

Keywords: DNA damage; DNA double-strand breaks; DNA repair; chromosome decatenation; genome instability; homologous recombination; non-homologous end-joining.

Figure 1

Repair of frank DSBs and seDSBs at collapsed replication forks. (A) Nucleases and ionizing radiation create frank, two-ended DSBs processed mainly by cNHEJ and HR regulated by factors that suppress resection (53BP1, RIF1) and those that promote resection (CtIP, MRN, DNA2, and EXO1), controlled by BRCA1. EEPD1 promotes resection through interactions with EXO1. Metnase promotes cNHEJ by methylating histone H3 (red symbols) in nucleosomes (grey ovals) near the DSB, and by promoting recruitment/retention of Ku and MRN. DNA-PKcs interacts with Ku and DNA ends to align ends and promote ligation by DNA ligase IV and other factors. Resected ends are repaired by HR by RAD51 loaded onto resected DNA, mediated by many factors including RPA and BRCA2. RAD51-ssDNA invades homologous duplex DNA and the end is extended (red, dashed arrows), and then released to pair with the second resected end. Gap filling and ligation completes accurate HR repair. (B) aNHEJ and SSA are backup repair pathways.  aNHEJ results in larger deletions as ends are aligned at 1-6 nt microhomologies (red rectangles) flanking the DSB exposed by limited resection.  3’ flaps are trimmed by ERCC1-XPF and Ligase III-XRCC1 and Pol θ complete repair that results in loss of one microhomology and intervening sequences.  SSA is analogous to aNHEJ but requires extensive resection to expose repeated sequences that anneal in a RAD52-dependent reaction.  SSA between linked repeats (shown) deletes one repeat and the intervening sequence; SSA between non-linked repeats results in translocations (not shown). (C) Forks stalled at blocking lesions can regress to a 4-way branched (chicken foot) structure similar to a Holiday junction (HJ). Extension of the leading nascent strand using the lagging nascent strand as template allows the leading strand to bypass the lesion in the leading template strand. The regressed fork can be restored to a functional fork by reverse branch migration, or by RAD51-mediated strand invasion beyond the blocking lesion. (D) Forks may collapse to seDSBs by encountering a single-strand nick, or blocked forks may be cleaved by MUS81 or EEPD1. Resection of the seDSB by EXO1 is promoted by both EEPD1 and Metnase, allowing RAD51-mediated HR to reestablish the fork. Metnase nuclease doesn’t cleave forks, but it may promote HR-mediated fork restart by processing late HR intermediates.

Figure 2

Structures and roles of replication stress nucleases. (A) Metnase is a fusion of SET and nuclease domains. Two S/TQ sites (potential PIKK targets) are indicated, along with the DDN nuclease motif. S508 is phosphorylated by Chk1. (B) Crystal structure of Metnase nuclease domains shown as a dimer (separated by dashed line), as solved by the Georgiadis lab (59); image from the Protein Data Bank Japan (60) using the Molmil molecular structure viewer (61). Positions of DDN core nuclease residues are indicated in dimer chain B by red dots. (C) EEPD1 has two helix-hairpin-helix (HhH) domains related to prokaryotic RuvA2, a component of RuvAB that mediates Holliday junction branch migration. Two potential PIKK target SQ sites are indicated. (D) Predicted EEPD1 structure showing HhH and nuclease domains with intervening non-structured regions; image from AlphaFold (62). (E) Summary of known functions of three replication stress nucleases. ND, not determined; +, promotes process; -, not involved in indicated process. See text for further details.