Although DNA replication is a tightly regulated process, cells are vulnerable to replication stress-induced genome instability, which is an enabling characteristic of tumourigenesis. Many chemotherapies work by interfering with cancer cell DNA replication. In response to replication stress, the replication fork is frequently remodelled (or reversed) into a 4-way chicken-foot-like structure which is protected from uncontrolled nucleolytic resection by a network of protective factors including the canonical tumour suppressors BRCA1 and BRCA2. Among the nucleases capable of acting at reversed forks is MRE11, which possesses both endonuclease and exonuclease activities and is a component of the MRN (MRE11, RAD50, NBS1) complex, which initiates Double-Strand Break (DSB) resection during DNA Double-Strand Break (DSB) repair. Our lab identified C5ORF45/MRE11-RAD50-NBS1 Interacting Protein (MRNIP) as a novel MRE11-interacting factor that limits nascent DNA degradation at stalled, reversed replication forks. However, the mechanisms underpinning fork protection by MRNIP are still unclear, and the further elucidation of MRNIP function is among the core aims of this study. MRNIP KO cells exhibit reduced levels of replication stress-induced MRE11 phosphorylation on Ser676/678. We demonstrate that expression of a phospho-mimetic MRE11 mutant prevents DNA degradation in MRNIP KO cells, suggesting that MRNIP-mediated MRE11 phosphorylation is crucial in preventing nascent DNA resection at reversed forks. In addition, we aimed to determine the role of MRNIP in the response to different chemotherapeutic drugs. Our findings indicate that cells lacking MRNIP are sensitive to the topoisomerase inhibitor Camptothecin but exhibit resistance to the chain terminating nucleoside analogue (CTNA) Gemcitabine. MRNIP was recently identified as being phosphorylated on Ser217, which is a potential non-canonical cyclin- dependent kinase 1 (CDK1) site. We find that Gemcitabine resistance in MRNIP KO cells requires Ser217, suggesting that MRNIP phosphorylation might provide a means to elicit cell cycle-specific control of MRE11. The function of this phosphorylation site appears context-dependent since alanine substitution of Ser217 did not affect MRE11-mediated nascent DNA degradation at HU-stalled forks.

Attempts to investigate nascent DNA degradation in response to Gemcitabine led to some unexpected but interesting results. We observed DNA degradation even in MRNIP-proficient cells treated with Gemcitabine, a process that seems to be independent of the action of the major nucleases commonly implicated at these sites.

We also examined MRNIP expression in a series of ovarian cancer cell (OC) lines. Loss of MRNIP protein and reduced mRNA levels were observed in certain High-Grade Ovarian Serous Adenocarcinoma cell lines, and MRNIP depletion led to different survival outcomes in response to different chemotherapeutic agents. We also identified CDK4 as a novel MRNIP interactor and found that CDK4 is stabilised in the absence of MRNIP, leading to elevated CDK4 levels in MRNIP KO cells. We also identify the E3 ligase Carboxy-terminus of Hsc70-interacting protein (CHIP) as a potential functional interactor and hypothesise that MRNIP limits the ability of CHIP to target and degrade CDK4.

In summary, this work reveals several novel phenotypes associated with MRNIP loss of function and adds depth to our mechanistic understanding of the functionality of a series of post-translational modifications of both MRE11 and MRNIP.