BLM and BRCA1-BARD1 coordinate complementary mechanisms of joint DNA molecule resolution.

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.

Repeated strand invasion and extensive branch migration are hallmarks of meiotic recombination.

Currently favored models for meiotic recombination posit that both noncrossover and crossover recombination are initiated by DNA double-strand breaks but form by different mechanisms: noncrossovers by synthesis-dependent strand annealing and crossovers by formation and resolution of double Holliday junctions centered around the break. This dual mechanism hypothesis predicts different hybrid DNA patterns in noncrossover and crossover recombinants. We show that these predictions are not upheld, by mapping with unprecedented resolution parental strand contributions to recombinants at a model locus. Instead, break repair in both noncrossovers and crossovers involves synthesis-dependent strand annealing, often with multiple rounds of strand invasion. Crossover-specific double Holliday junction formation occurs via processes involving branch migration as an integral feature, one that can be separated from repair of the break itself. These findings reveal meiotic recombination to be a highly dynamic process and prompt a new view of the relationship between crossover and noncrossover recombination.

Regulated Proteolysis of MutSγ Controls Meiotic Crossing Over.

Crossover recombination is essential for accurate chromosome segregation during meiosis. The MutSγ complex, Msh4-Msh5, facilitates crossing over by binding and stabilizing nascent recombination intermediates. We show that these activities are governed by regulated proteolysis. MutSγ is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable by directly targeting proteasomal degradation. Activation of MutSγ requires the Dbf4-dependent kinase Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Genetic requirements for Msh4 phosphorylation indicate that DDK targets MutSγ only after it has bound to nascent joint molecules (JMs) in the context of synapsing chromosomes. Overexpression studies confirm that the steady-state level of Msh4, not phosphorylation per se, is the critical determinant for crossing over. At the DNA level, Msh4 phosphorylation enables the formation and crossover-biased resolution of double-Holliday Junction intermediates. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossing over.

Network Rewiring of Homologous Recombination Enzymes during Mitotic Proliferation and Meiosis.

Homologous recombination (HR) is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the recombination machinery to avoid (mitosis) or enforce (meiosis) the formation of reciprocal exchanges-crossovers-between recombining chromosomes. To obtain molecular insight into how crossover control is achieved, we affinity purified 7 DNA-processing enzymes that channel HR intermediates into crossovers or noncrossovers from vegetative cells or cells undergoing meiosis. Using mass spectrometry, we provide a global characterization of their composition and reveal mitosis- and meiosis-specific modules in the interaction networks. Functional analyses of meiosis-specific interactors of MutLγ-Exo1 identified Rtk1, Caf120, and Chd1 as regulators of crossing-over. Chd1, which transiently associates with Exo1 at the prophase-to-metaphase I transition, enables the formation of MutLγ-dependent crossovers through its conserved ability to bind and displace nucleosomes. Thus, rewiring of the HR network, coupled to chromatin remodeling, promotes context-specific control of the recombination outcome.

Mechanistic View and Genetic Control of DNA Recombination during Meiosis.

Meiotic recombination is essential for fertility and allelic shuffling. Canonical recombination models fail to capture the observed complexity of meiotic recombinants. Here, by combining genome-wide meiotic heteroduplex DNA patterns with meiotic DNA double-strand break (DSB) sites, we show that part of this complexity results from frequent template switching during synthesis-dependent strand annealing that yields noncrossovers and from branch migration of double Holliday junction (dHJ)-containing intermediates that mainly yield crossovers. This complexity also results from asymmetric positioning of crossover intermediates relative to the initiating DSB and Msh2-independent conversions promoted by the suspected dHJ resolvase Mlh1-3 as well as Exo1 and Sgs1. Finally, we show that dHJ resolution is biased toward cleavage of the pair of strands containing newly synthesized DNA near the junctions and that this bias can be decoupled from the crossover-biased dHJ resolution. These properties are likely conserved in eukaryotes containing ZMM proteins, which includes mammals.

Bacteriophage lambda site-specific recombination.

The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage-encoded Int and Xis proteins and the host-encoded integration host factor and factor for inversion stimulation proteins. Structures of higher-order protein-DNA complexes of the four-way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site-specific recombination, with an emphasis on key studies completed over the last decade.

GEN1 as a risk factor for human congenital anomalies of the kidney and urinary tract.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are prevalent birth defects. Although pathogenic CAKUT genes are known, they are insufficient to reveal the causes for all patients. Our previous studies indicated GEN1 as a pathogenic gene of CAKUT in mice, and this study further investigated the correlation between GEN1 and human CAKUT. METHODS: In this study, DNA from 910 individuals with CAKUT was collected; 26 GEN1 rare variants were identified, and two GEN1 (missense) variants in a non-CAKUT group were found. Mainly due to the stability results of the predicted mutant on the website, in vitro, 10 variants (eight CAKUT, two non-CAKUT) were selected to verify mutant protein stability. In addition, mainly based on the division of the mutation site located in the functional region of the GEN1 protein, 8 variants (six CAKUT, two non-CAKUT) were selected to verify enzymatic hydrolysis, and the splice variant GEN1 (c.1071 + 3(IVS10) A > G) was selected to verify shear ability. Based on the results of in vitro experiments and higher frequency, three sites with the most significant functional change were selected to build mouse models. RESULTS: Protein stability changed in six variants in the CAKUT group. Based on electrophoretic mobility shift assay of eight variants (six CAKUT, two non-CAKUT), the enzymatic hydrolysis and DNA-binding abilities of mutant proteins were impaired in the CAKUT group. The most serious functional damage was observed in the Gen1 variant that produced a truncated protein. A mini-gene splicing assay showed that the variant GEN1 (c.1071 + 3(IVS10) A > G) in the CAKUT group significantly affected splicing function. An abnormal exon10 was detected in the mini-gene splicing assay. Point-mutant mouse strains were constructed (Gen1: c.1068 + 3 A > G, p.R400X, and p.T105R) based on the variant frequency in the CAKUT group and functional impairment in vitro study and CAKUT phenotypes were replicated in each. CONCLUSION: Overall, our findings indicated GEN1 as a risk factor for human CAKUT.

The SMX DNA Repair Tri-nuclease.

The efficient removal of replication and recombination intermediates is essential for the maintenance of genome stability. Resolution of these potentially toxic structures requires the MUS81-EME1 endonuclease, which is activated at prometaphase by formation of the SMX tri-nuclease containing three DNA repair structure-selective endonucleases: SLX1-SLX4, MUS81-EME1, and XPF-ERCC1. Here we show that SMX tri-nuclease is more active than the three individual nucleases, efficiently cleaving replication forks and recombination intermediates. Within SMX, SLX4 co-ordinates the SLX1 and MUS81-EME1 nucleases for Holliday junction resolution, in a reaction stimulated by XPF-ERCC1. SMX formation activates MUS81-EME1 for replication fork and flap structure cleavage by relaxing substrate specificity. Activation involves MUS81's conserved N-terminal HhH domain, which mediates incision site selection and SLX4 binding. Cell cycle-dependent formation and activation of this tri-nuclease complex provides a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.

Analysis of non-canonical three- and four-way DNA junctions.

The development of compounds that can selectively bind with non-canonical DNA structures has expanded in recent years. Junction DNA, including three-way junctions (3WJs) and four-way Holliday junctions (HJs), offer an intriguing target for developmental therapeutics as both 3WJs and HJs are involved in DNA replication and repair processes. However, there are a limited number of assays available for the analysis of junction DNA binding. Here, we describe the design and execution of multiplex fluorescent polyacrylamide gel electrophoresis (PAGE) and microscale thermophoresis (MST) assays that enable evaluation of junction-binding compounds. Two well characterised junction-binding compounds-a C6 linked bis-acridine ligand and an iron(II)-bound peptide helicate, which recognise HJs and 3WJs, respectively-were employed as probes for both MST and PAGE experiments. The multiplex PAGE assay expands beyond previously reported fluorescent PAGE as it uses four individual fluorophores that can be combined to visualise single-strands, pseudo-duplexes, and junction DNA present during 3WJ and HJ formation. The use of MST to identify the binding affinity of junction binding agents is, to our knowledge, first reported example of this technique. The combined use of PAGE and MST provides complementary results for the visualisation of 3WJ and HJ formation and the direct binding affinity (Kd and EC50) of these agents. These assays can be used to aid the discovery and design of new therapeutics targeting non-canonical nucleic acid structures.

Inhibition of MAPK/ERK pathway activation rescues congenital anomalies of the kidney and urinary tract (CAKUT) in Robo2PB/+ Gen1PB/+ mice.

Congenital anomalies of the kidney and urinary tract (CAKUT) have been attributed to genetic and environmental factors. However, monogenic and copy number variations cannot sufficiently explain the cause of the majority of CAKUT cases. Multiple genes through various modes of inheritance may lead to CAKUT pathogenesis. We previously showed that Robo2 and Gen1 coregulated the germination of ureteral buds (UB), significantly increasing CAKUT incidence. Furthermore, MAPK/ERK pathway activation is the central mechanism of these two genes. Thus, we explored the effect of the MAPK/ERK inhibitor U0126 in the CAKUT phenotype in Robo2PB/+Gen1PB/+ mice. Intraperitoneal injection of U0126 during pregnancy prevented the development of the CAKUT phenotype in Robo2PB/+Gen1PB/+ mice. Additionally, a single dose of 30 mg/kg U0126 on day 10.5 embryos (E10.5) was most effective for reducing CAKUT incidence and ectopic UB outgrowth in Robo2PB/+Gen1PB/+ mice. Furthermore, embryonic kidney mesenchymal levels of p-ERK were significantly decreased on day E11.5 after U0126 treatment, along with decreased cell proliferation index PHH3 and ETV5 expression. Collectively, Gen1 and Robo2 exacerbated the CAKUT phenotype in Robo2PB/+Gen1PB/+ mice through the MAPK/ERK pathway, increasing proliferation and ectopic UB outgrowth.

Method to generate Holliday junction recombination intermediates via RecA-mediated four-strand exchange.

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts.

The scientific revolution that unraveled the astonishing DNA repair capacity of the Deinococcaceae: 40 years on.

The family Deinococcaceae exhibits exceptional radiation resistance and possesses all the necessary traits for surviving in radiation-exposed environments. Their survival strategy involves the coupling of metabolic and DNA repair functions, resulting in an extraordinarily efficient homologous repair of DNA double-strand breaks (DSBs) caused by radiation or desiccation. The keys to their survival lie in the hyperaccumulation of manganous (Mn2+)-metabolite antioxidants that protect their DNA repair proteins under extreme oxidative stress and the persistent structural linkage by Holliday junctions of their multiple genome copies per cell that facilitates DSB repair. This coupling of metabolic and DNA repair functions has made polyploid Deinococcus bacteria a useful tool in environmental biotechnology, radiobiology, aging, and planetary protection. The review highlights the groundbreaking contributions of the late Robert G.E. Murray to the field of Deinococcus research and the emergent paradigm-shifting discoveries that revolutionized our understanding of radiation survivability and oxidative stress defense, demonstrating that the proteome, rather than the genome, is the primary target responsible for survivability. These discoveries have led to the commercial development of irradiated vaccines using Deinococcus Mn-peptide antioxidants and have significant implications for various fields.

Effects of Holliday Junction-Recognition Protein-Mediated C-Jun N-Terminal Kinase/ Signal Transducer and Activator of Transcription 3 Signaling Pathway on Cell Proliferation, Cell Cycle and Cell Apoptosis in Bladder Urothelial Carcinoma.

The Holliday Junction-Recognition Protein (HJURP) was upregulated in several tumors, which was associated with poor outcome. This study investigated the effects of the HJURP-mediated c-Jun N-terminal kinase (JNK)/ signal transducer and activator of transcription 3 (STAT3) pathway on bladder urothelial carcinoma (BLUC). Online databases were used to analyze HJURP expression in BLUC and the correlation of HJURP to JNK1 [mitogen-activated protein kinase 8 (MAPK8)], JNK2 (MAPK9), STAT3, marker of proliferation Ki-67 (MKI67), proliferating cell nuclear antigen (PCNA), cyclin dependent kinase 2 (CDK2), CDK4 and CDK6. HJURP expression was detected in BLUC cells and human normal primary bladder epithelial cells (BdECs). BLUC cells were treated with HJURP lentivirus activation /shRNA lentivirus particles or JNK inhibitor SP600125. HJURP was upregulated in BLUC tissues and correlated with poor prognosis of patients (all P < 0.05). HJURP in tumor positively correlated with MAPK8 (R = 0.30), MAPK9 (R = 0.30), STAT3 (R = 0.15), MKI67 (R = 0.60), PCNA (R = 0.46), CDK2 (R = 0.39), CDK4 (R = 0.24) and CDK6 (R = 0.21). The JNK inhibitor SP600125 decreased p-JNK/JNK and p-STAT3/STAT3 in BLUC cells, which was reversed by HJURP overexpression (P < 0.05). The HJURP-mediated JNK/STAT3 pathway promoted BLUC cell proliferation and inhibited cell apoptosis (P < 0.05). HJURP reversed the arrested G0/G1 phase of BLUC cells by SP600125. HJURP acted as an oncogene to regulate BLUC cell proliferation, apoptosis and the cell cycle by mediating the JNK/STAT3 pathway. Therefore, HJURP targeting might be an attractive novel therapeutic target for early diagnosis and treatment in BLUC.

Junctions in DNA: underexplored targets for therapeutic intervention.

DNA has been a key target for cancer therapy, with a range of compounds able to bind and either impair its processing or induce damage. Targeting DNA with small molecules in a truly sequence specific way, to impair gene specific processes, remains out of reach. The ability of DNA to assume different structures from the classical double helix allows access to more specific ligand binding modes and, potentially, to new avenues of treatment. In this review, we illustrate the small molecules that have been reported to bind to three- and four-way junctions.

Identification of small-molecule inhibitors of the DNA repair proteins RuvAB from Pseudomonas aeruginosa.

The Holliday junction (HJ) branch migrator RuvAB complex plays a fundamental role during homologous recombination and DNA damage repair, and therefore, is an attractive target for the treatment of bacterial pathogens. Pseudomonas aeruginosa (P. aeruginosa, Pa) is one of the most common clinical opportunistic bacterial pathogens, which can cause a series of life-threatening acute or chronic infections. Here, we performed a high throughput small-molecule screening targeting PaRuvAB using the FRET-based HJ branch migration assay. We identified that corilagin, bardoxolone methyl (BM) and 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SKQ1) could efficiently inhibit the branch migration activity of PaRuvAB, with IC50 values of 0.40 ± 0.04 µM, 0.38 ± 0.05 µM and 4.64 ± 0.27 µM, respectively. Further biochemical and molecular docking analyses demonstrated that corilagin directly bound to PaRuvB at the ATPase domain, and thus prevented ATP hydrolysis. In contrast, BM and SKQ1 acted through blocking the interactions between PaRuvA and HJ DNA. Finally, these compounds were shown to increase the susceptibility of P. aeruginosa to UV-C irradiation. Our work, for the first time, reports the small-molecule inhibitors of RuvA and RuvB from any species, providing valuable chemical tools to dissect the functional role of each individual protein in vivo.

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli, nontypeable Haemophilus influenzae, and Staphylococcus epidermidis Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.

Roles of SLX1-SLX4, MUS81-EME1, and GEN1 in avoiding genome instability and mitotic catastrophe.

The resolution of recombination intermediates containing Holliday junctions (HJs) is critical for genome maintenance and proper chromosome segregation. Three pathways for HJ processing exist in human cells and involve the following enzymes/complexes: BLM-TopoIIIα-RMI1-RMI2 (BTR complex), SLX1-SLX4-MUS81-EME1 (SLX-MUS complex), and GEN1. Cycling cells preferentially use the BTR complex for the removal of double HJs in S phase, with SLX-MUS and GEN1 acting at temporally distinct phases of the cell cycle. Cells lacking SLX-MUS and GEN1 exhibit chromosome missegregation, micronucleus formation, and elevated levels of 53BP1-positive G1 nuclear bodies, suggesting that defects in chromosome segregation lead to the transmission of extensive DNA damage to daughter cells. In addition, however, we found that the effects of SLX4, MUS81, and GEN1 depletion extend beyond mitosis, since genome instability is observed throughout all phases of the cell cycle. This is exemplified in the form of impaired replication fork movement and S-phase progression, endogenous checkpoint activation, chromosome segmentation, and multinucleation. In contrast to SLX4, SLX1, the nuclease subunit of the SLX1-SLX4 structure-selective nuclease, plays no role in the replication-related phenotypes associated with SLX4/MUS81 and GEN1 depletion. These observations demonstrate that the SLX1-SLX4 nuclease and the SLX4 scaffold play divergent roles in the maintenance of genome integrity in human cells.

Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius.

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

Integration Host Factor Binds DNA Holliday Junctions.

Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.

DNA Holliday Junction: History, Regulation and Bioactivity.

DNA Holliday junction (HJ) is a four-way stranded DNA intermediate that formed in replication fork regression, homology-dependent repair and mitosis, performing a significant role in genomic stability. Failure to remove HJ can induce an acceptable replication fork stalling and DNA damage in normal cells, leading to a serious chromosomal aberration and even cell death in HJ nuclease-deficient tumor cells. Thus, HJ is becoming an attractive target in cancer therapy. However, the development of HJ-targeting ligand faces great challenges because of flexile cavities on the center of HJs. This review introduces the discovery history of HJ, elucidates the formation and dissociation procedures of HJ in corresponding bio-events, emphasizes the importance of prompt HJ-removing in genome stability, and summarizes recent advances in HJ-based ligand discovery. Our review indicate that target HJ is a promising approach in oncotherapy.