SLX4 dampens MutSα-dependent mismatch repair

ABSTRACTThe tumour suppressor SLX4 plays multiple roles in the maintenance of genome stability, acting as a scaffold for structure-specific endonucleases and other DNA repair proteins. It directly interacts with the mismatch repair (MMR) protein MSH2 but the significance of this interaction remained unknown until recent findings showing that MutSβ (MSH2-MSH3) stimulates in vitro the SLX4-dependent Holliday junction resolvase activity. Here, we characterize the mode of interaction between SLX4 and MSH2, which relies on an MSH2-interacting peptide (SHIP box) that drives interaction of SLX4 with both MutSβ and MutSα (MSH2-MSH6). While we show that this MSH2 binding domain is dispensable for the well-established role of SLX4 in interstrand crosslink repair, we find that it mediates inhibition of MutSα-dependent MMR by SLX4, unravelling an unanticipated function of SLX4.

Analysis of Adaptive Olaparib Resistance Effects on Cisplatin Sensitivity in Triple Negative Breast Cancer Cells

Poly-(ADP)-ribose polymerase inhibitors (PARPi) and platinum-based drugs are promising therapies for triple negative breast cancers (TNBC) with BRCA1 or BRCA2 loss. PARPi(s) show better efficacies when combined with platinum-based therapy, however, acquisition of PARPi resistance has been linked with co-resistance to platinum-based drugs. Here, we show that TNBCs with constitutively hyperactivated PARP-1 display greater tolerances for the PARPi olaparib and cisplatin, and respond synergistically to olaparib/cisplatin combinations with increased cytotoxicity. Regardless of BRCA1 and PARP-1 activity status, upon gaining olaparib resistance (OlaR), OlaR MDA-MB-468 (BRCA1 wild-type) and SUM1315 (BRCA1 mutant) TNBC cells retain cisplatin sensitivities of their isogenic parental counterparts. OlaR TNBC cells express decreased levels of PARP-1 and Pol η, a translesion-synthesis polymerase important in platinum-induced interstrand crosslink repair. Although native RAD51 recombinase levels are unaffected, anti-RAD51 immunoreactive low molecular weight sbands are exclusively detected in OlaR cells. Despite normal BRCA1, RAD51 foci formation/recruitment to double-strand breaks are impaired in OlaR MDA-MB-468 cells, suggesting homologous-recombination impairment. RNA-seq and pathway analysis of cisplatin-affected genes revealed enrichment of G2/M cell cycle regulation and DNA repair pathways in parental and OlaR MDA-MB-468 cells whereas parental and OlaR SUM1315 cells showed enrichment of inflammatory stress response pathways associated with TNFR1/2, TWEAK and IL-17 signaling. These data show that TNBC models with wild type versus mutant BRCA1 exhibit differences in CDDP-induced cellular response pathways, however, the CDDP-induced signaling responses remain stable across the isogenic models of OlaR from the same lineage. These data also show that adaptive OlaR does not automatically promote cisplatin resistance, implicating the potential benefit of platinum-based therapy for OlaR TNBCs.

FANCD2 directly inhibits DNA2 nuclease at stalled replication forks and acts as a RAD51 mediator in strand exchange

FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea induced stalled forks. The mechanisms of fork protection are not well studied. Here, we purified FANCD2 to study how FANCD2 regulates DNA resection at stalled forks. In vitro, we showed that FANCD2 inhibits fork degradation in two ways: 1) it inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit various nucleases, including DNA2. More unexpectedly, FANCD2 acts as a RAD51 mediator to stimulate the strand exchange activity of RAD51, and does so by enhancing ssDNA binding of RAD51. Our work biochemically explains mechanisms by which FANCD2 protects stalled forks and further provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator.

Probing the Binding Requirements of Modified Nucleosides with the DNA Nuclease SNM1A

SNM1A is a nuclease that is implicated in DNA interstrand crosslink repair and, as such, its inhibition is of interest for overcoming resistance to chemotherapeutic crosslinking agents. However, the number and identity of the metal ion(s) in the active site of SNM1A are still unconfirmed, and only a limited number of inhibitors have been reported to date. Herein, we report the synthesis and evaluation of a family of malonate-based modified nucleosides to investigate the optimal positioning of metal-binding groups in nucleoside-derived inhibitors for SNM1A. These compounds include ester, carboxylate and hydroxamic acid malonate derivatives which were installed in the 5′-position or 3′-position of thymidine or as a linkage between two nucleosides. Evaluation as inhibitors of recombinant SNM1A showed that nine of the twelve compounds tested had an inhibitory effect at 1 mM concentration. The most potent compound contains a hydroxamic acid malonate group at the 5′-position. Overall, our studies advance the understanding of requirements for nucleoside-derived inhibitors for SNM1A and indicate that groups containing a negatively charged group in close proximity to a metal chelator, such as hydroxamic acid malonates, are promising structures in the design of inhibitors.

The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair

DNA interstrand crosslink (ICL) repair requires a complex network of DNA damage response pathways. Removal of the ICL lesions is vital, as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principal mechanism for ICL repair in metazoans and is coupled to DNA replication. In Saccharomyces cerevisiae, a vestigial FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease, which is hypothesized to use its exonuclease activity to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked enzyme RecQ-like helicase 4 (RECQL4), as a component of Pso2-mediated ICL repair. Here, using genetic, biochemical, and biophysical approaches, including single-molecule FRET (smFRET)– and gel-based nuclease assays, we show that Hrq1 stimulates the Pso2 nuclease through a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulated Pso2 translesion nuclease activity through a site-specific ICL in vitro. We noted that stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and genetic and biochemical data suggest that Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these detrimental DNA lesions.

HSF2BP negatively regulates homologous recombination in DNA interstrand crosslink repair

The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability and DNA interstrand crosslink (ICL) repair in vertebrates. We show that ectopic production of HSF2BP, a BRCA2-interacting protein required for meiotic HR during mouse spermatogenesis, in non-germline human cells acutely sensitize them to ICL-inducing agents (mitomycin C and cisplatin) and PARP inhibitors, resulting in a phenotype characteristic of cells from Fanconi anemia (FA) patients. We biochemically recapitulate the suppression of ICL repair and establish that excess HSF2BP compromises HR by triggering the removal of BRCA2 from the ICL site and thereby preventing the loading of RAD51. This establishes ectopic expression of a wild-type meiotic protein in the absence of any other protein-coding mutations as a new mechanism that can lead to an FA-like cellular phenotype. Naturally occurring elevated production of HSF2BP in tumors may be a source of cancer-promoting genomic instability and also a targetable vulnerability.