Xrs2/NBS1 promote end-bridging activity of the MRE11-RAD50 complex.

DNA double strand breaks (DSBs) can be detrimental to the cell and need to be efficiently repaired. A first step in DSB repair is to bring the free ends in close proximity to enable ligation by non-homologous end-joining (NHEJ), while the more precise, but less available, repair by homologous recombination (HR) requires close proximity of a sister chromatid. The human MRE11-RAD50-NBS1 (MRN) complex, Mre11-Rad50-Xrs2 (MRX) in yeast, is involved in both repair pathways. Here we use nanofluidic channels to study, on the single DNA molecule level, how MRN, MRX and their constituents interact with long DNA and promote DNA bridging. Nanofluidics is a suitable method to study reactions on DNA ends since no anchoring of the DNA end(s) is required. We demonstrate that NBS1 and Xrs2 play important, but differing, roles in the DNA tethering by MRN and MRX. NBS1 promotes DNA bridging by MRN consistent with tethering of a repair template. MRX shows a "synapsis-like" DNA end-bridging, stimulated by the Xrs2 subunit. Our results highlight the different ways MRN and MRX bridge DNA, and the results are in agreement with their key roles in HR and NHEJ, respectively, and contribute to the understanding of the roles of NBS1 and Xrs2 in DSB repair.

PLK1 regulates CtIP and DNA2 interplay in long-range DNA end resection.

DNA double-strand break (DSB) repair is initiated by DNA end resection. CtIP acts in short-range resection to stimulate MRE11-RAD50-NBS1 (MRN) to endonucleolytically cleave 5'-terminated DNA to bypass protein blocks. CtIP also promotes the DNA2 helicase-nuclease to accelerate long-range resection downstream from MRN. Here, using AlphaFold2, we identified CtIP-F728E-Y736E as a separation-of-function mutant that is still proficient in conjunction with MRN but is not able to stimulate ssDNA degradation by DNA2. Accordingly, CtIP-F728E-Y736E impairs physical interaction with DNA2. Cellular assays revealed that CtIP-F728E-Y736E cells exhibit reduced DSB-dependent chromatin-bound RPA, impaired long-range resection, and increased sensitivity to DSB-inducing drugs. Previously, CtIP was shown to be targeted by PLK1 to inhibit long-range resection, yet the underlying mechanism was unclear. We show that the DNA2-interacting region in CtIP includes the PLK1 target site at S723. The integrity of S723 in CtIP is necessary for the stimulation of DNA2, and phosphorylation of CtIP by PLK1 in vitro is consequently inhibitory, explaining why PLK1 restricts long-range resection. Our data support a model in which CDK-dependent phosphorylation of CtIP activates resection by MRN in S phase, and PLK1-mediated phosphorylation of CtIP disrupts CtIP stimulation of DNA2 to attenuate long-range resection later at G2/M.

WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures.

The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long-term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1-SLX4 structure-specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2-MSH6), MutSß (MSH2-MSH3), and MutLα (MLH1-PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic-lethal interaction with promising applications in precision cancer therapy.

Double-stranded DNA binding function of RAD51 in DNA protection and its regulation by BRCA2.

RAD51 and the breast cancer suppressor BRCA2 have critical functions in DNA double-strand (dsDNA) break repair by homologous recombination and the protection of newly replicated DNA from nucleolytic degradation. The recombination function of RAD51 requires its binding to single-stranded DNA (ssDNA), whereas binding to dsDNA is inhibitory. Using reconstituted MRE11-, EXO1-, and DNA2-dependent nuclease reactions, we show that the protective function of RAD51 unexpectedly depends on its binding to dsDNA. The BRC4 repeat of BRCA2 abrogates RAD51 binding to dsDNA and accordingly impairs the function of RAD51 in protection. The BRCA2 C-terminal RAD51-binding segment (TR2) acts in a dominant manner to overcome the effect of BRC4. Mechanistically, TR2 stabilizes RAD51 binding to dsDNA, even in the presence of BRC4, promoting DNA protection. Our data suggest that RAD51's dsDNA-binding capacity may have evolved together with its function in replication fork protection and provide a mechanistic basis for the DNA-protection function of BRCA2.

The CDK1-TOPBP1-PLK1 axis regulates the Bloom's syndrome helicase BLM to suppress crossover recombination in somatic cells.

Bloom's syndrome is caused by inactivation of the BLM helicase, which functions with TOP3A and RMI1-2 (BTR complex) to dissolve recombination intermediates and avoid somatic crossing-over. We show here that crossover avoidance by BTR further requires the activity of cyclin-dependent kinase-1 (CDK1), Polo-like kinase-1 (PLK1), and the DDR mediator protein TOPBP1, which act in the same pathway. Mechanistically, CDK1 phosphorylates BLM and TOPBP1 and promotes the interaction of both proteins with PLK1. This is amplified by the ability of TOPBP1 to facilitate phosphorylation of BLM at sites that stimulate both BLM-PLK1 and BLM-TOPBP1 binding, creating a positive feedback loop that drives rapid BLM phosphorylation at the G2-M transition. In vitro, BLM phosphorylation by CDK/PLK1/TOPBP1 stimulates the dissolution of topologically linked DNA intermediates by BLM-TOP3A. Thus, we propose that the CDK1-TOPBP1-PLK1 axis enhances BTR-mediated dissolution of recombination intermediates late in the cell cycle to suppress crossover recombination and curtail genomic instability.

A Disease-Causing Single Amino Acid Deletion in the Coiled-Coil Domain of RAD50 Impairs MRE11 Complex Functions in Yeast and Humans.

The MRE11-RAD50-NBS1 complex plays a central role in response to DNA double-strand breaks. Here, we identify a patient with bone marrow failure and developmental defects caused by biallelic RAD50 mutations. One of the mutations creates a null allele, whereas the other (RAD50E1035Δ) leads to the loss of a single residue in the heptad repeats within the RAD50 coiled-coil domain. This mutation represents a human RAD50 separation-of-function mutation that impairs DNA repair, DNA replication, and DNA end resection without affecting ATM-dependent DNA damage response. Purified recombinant proteins indicate that RAD50E1035Δ impairs MRE11 nuclease activity. The corresponding mutation in Saccharomyces cerevisiae causes severe thermosensitive defects in both DNA repair and Tel1ATM-dependent signaling. These findings demonstrate that a minor heptad break in the RAD50 coiled coil suffices to impede MRE11 complex functions in human and yeast. Furthermore, these results emphasize the importance of the RAD50 coiled coil to regulate MRE11-dependent DNA end resection in humans.

Phosphorylated CtIP bridges DNA to promote annealing of broken ends.

The early steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The roles of these proteins in nucleolytic DSB resection are well characterized, but their role in bridging the DNA ends for efficient and correct repair is much less explored. Here we study the binding of phosphorylated CtIP, which promotes the endonuclease activity of MRN, to single long (∼50 kb) DNA molecules using nanofluidic channels and compare it to the yeast homolog Sae2. CtIP bridges DNA in a manner that depends on the oligomeric state of the protein, and truncated mutants demonstrate that the bridging depends on CtIP regions distinct from those that stimulate the nuclease activity of MRN. Sae2 is a much smaller protein than CtIP, and its bridging is significantly less efficient. Our results demonstrate that the nuclease cofactor and structural functions of CtIP may depend on the same protein population, which may be crucial for CtIP functions in both homologous recombination and microhomology-mediated end-joining.

The iron-sulphur cluster in human DNA2 is required for all biochemical activities of DNA2.

The nuclease/helicase DNA2 plays important roles in DNA replication, repair and processing of stalled replication forks. DNA2 contains an iron-sulphur (FeS) cluster, conserved in eukaryotes and in a related bacterial nuclease. FeS clusters in DNA maintenance proteins are required for structural integrity and/or act as redox-sensors. Here, we demonstrate that loss of the FeS cluster affects binding of human DNA2 to specific DNA substrates, likely through a conformational change that distorts the central DNA binding tunnel. Moreover, we show that the FeS cluster is required for DNA2's nuclease, helicase and ATPase activities. Our data also establish that oxidation of DNA2 impairs DNA binding in vitro, an effect that is reversible upon reduction. Unexpectedly, though, this redox-regulation is independent of the presence of the FeS cluster. Together, our study establishes an important structural role for the FeS cluster in human DNA2 and discovers a redox-regulatory mechanism to control DNA binding.

Phosphorylation of the RecQ Helicase Sgs1/BLM Controls Its DNA Unwinding Activity during Meiosis and Mitosis.

The Bloom's helicase ortholog, Sgs1, orchestrates the formation and disengagement of recombination intermediates to enable controlled crossing-over during meiotic and mitotic DNA repair. Whether its enzymatic activity is temporally regulated to implement formation of noncrossovers prior to the activation of crossover-nucleases is unknown. Here, we show that, akin to the Mus81-Mms4, Yen1, and MutLγ-Exo1 nucleases, Sgs1 helicase function is under cell-cycle control through the actions of CDK and Cdc5 kinases. Notably, however, whereas CDK and Cdc5 unleash nuclease function during M phase, they act in concert to stimulate Sgs1 activity during S phase/prophase I. Mechanistically, CDK-mediated phosphorylation enhances the velocity and processivity of Sgs1, which stimulates DNA unwinding in vitro and joint molecule processing in vivo. Subsequent hyper-phosphorylation by Cdc5 appears to reduce the activity of Sgs1, while activating Mus81-Mms4 and MutLγ-Exo1. These findings suggest a concerted mechanism driving orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.

CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection.

To repair a DNA double-strand break by homologous recombination, 5'-terminated DNA strands must first be resected to reveal 3'-overhangs. This process is initiated by a short-range resection catalyzed by MRE11-RAD50-NBS1 (MRN) stimulated by CtIP, which is followed by a long-range step involving EXO1 or DNA2 nuclease. DNA2 is a bifunctional enzyme that contains both single-stranded DNA (ssDNA)-specific nuclease and motor activities. Upon DNA unwinding by Bloom (BLM) or Werner (WRN) helicase, RPA directs the DNA2 nuclease to degrade the 5'-strand. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single-molecule biochemistry, we show that CtIP also dramatically stimulates the adenosine 5'-triphosphate (ATP) hydrolysis-driven motor activity of DNA2 involved in the long-range resection step. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. Accordingly, the stimulatory effect of CtIP is only observed with wild-type DNA2, but not the helicase-deficient variant. Similarly to the function of CtIP to promote MRN, also the DNA2 stimulatory effect is facilitated by CtIP phosphorylation. The domain of CtIP required to promote DNA2 is located in the central region lacking in lower eukaryotes and is fully separable from domains involved in the stimulation of MRN. These results establish how CtIP couples both MRE11-dependent short-range and DNA2-dependent long-range resection and define the involvement of the motor activity of DNA2 in this process. Our data might help explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.

The internal region of CtIP negatively regulates DNA end resection.

DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of MRE11-RAD50-NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM-DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region in DNA end resection. We found that residues 350-600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN-CtIP-dependent resection activity for repair. This suggested that the internal CtIP region, further mapped to residues 550-600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta.

Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment.