Double-negative B cells and DNASE1L3 colocalise with microbiota in gut-associated lymphoid tissue.

Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the subepithelial dome. The initial response to antigens and how bacteria are handled is incompletely understood. By iterative application of spatial transcriptomics and multiplexed single-cell technologies, we identify that the double negative 2 subset of B cells, previously associated with autoimmune diseases, is present in the subepithelial dome in health. We show that in this location double negative 2 B cells interact with dendritic cells co-expressing the lupus autoantigens DNASE1L3 and C1q and microbicides. We observe that in humans, but not in mice, dendritic cells expressing DNASE1L3 are associated with sampled bacteria but not DNA derived from apoptotic cells. We propose that fundamental features of autoimmune diseases are microbiota-associated, interacting components of normal intestinal immunity.

The Terminase Complex of Each Human Herpesvirus.

The human herpesviruses (HHVs) are classified into the following three subfamilies: Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. These HHVs have distinct pathological features, while containing a highly conserved viral replication pathway. Among HHVs, the basic viral particle structure and the sequential processes of viral replication are nearly identical. In particular, the capsid formation mechanism has been proposed to be highly similar among herpesviruses, because the viral capsid-organizing proteins are highly conserved at the structural and functional levels. Herpesviruses form capsids containing the viral genome in the nucleus of infected cells during the lytic phase, and release infectious virus (i.e., virions) to the cell exterior. In the capsid formation process, a single-unit-length viral genome is encapsidated into a preformed capsid. The single-unit-length viral genome is produced by cleavage from a viral genome precursor in which multiple unit-length viral genomes are tandemly linked. This encapsidation and cleavage is carried out by the terminase complex, which is composed of viral proteins. Since the terminase complex-mediated encapsidation and cleavage is a virus-specific mechanism that does not exist in humans, it may be an excellent inhibitory target for anti-viral drugs with high virus specificity. This review provides an overview of the functions of the terminase complexes of HHVs.

BRCA1 safeguards genome integrity by activating chromosome asynapsis checkpoint to eliminate recombination-defective oocytes.

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.

Deciphering the Substrate Specificity Reveals that CRISPR-Cas12a Is a Bifunctional Enzyme with Both Endo- and Exonuclease Activities.

The class 2 CRISPR-Cas9 and CRISPR-Cas12a systems, originally described as adaptive immune systems of bacteria and archaea, have emerged as versatile tools for genome-editing, with applications in biotechnology and medicine. However, significantly less is known about their substrate specificity, but such knowledge may provide instructive insights into their off-target cleavage and previously unrecognized mechanism of action. Here, we document that the Acidaminococcus sp. Cas12a (AsCas12a) binds preferentially, and independently of crRNA, to a suite of branched DNA structures, such as the Holliday junction (HJ), replication fork and D-loops, compared with single- or double-stranded DNA, and promotes their degradation. Further, our study revealed that AsCas12a binds to the HJ, specifically at the crossover region, protects it from DNase I cleavage and renders a pair of thymine residues in the HJ homologous core hypersensitive to KMnO4 oxidation, suggesting DNA melting and/or distortion. Notably, these structural changes enabled AsCas12a to resolve HJ into nonligatable intermediates, and subsequently their complete degradation. We further demonstrate that crRNA impedes HJ cleavage by AsCas12a, and that of Lachnospiraceae bacterium Cas12a, without affecting their DNA-binding ability. We identified a separation-of-function variant, which uncouples DNA-binding and DNA cleavage activities of AsCas12a. Importantly, we found robust evidence that AsCas12a endonuclease also has 3'-to-5' and 5'-to-3' exonuclease activity, and that these two activities synergistically promote degradation of DNA, yielding di- and mononucleotides. Collectively, this study significantly advances knowledge about the substrate specificity of AsCas12a and provides important insights into the degradation of different types of DNA substrates.

Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m6A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m6A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.

High-Throughput µPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil.

The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (µPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site, and the arsM abundance ranged from 1.05 × 106 to 6.49 × 107 copies/g; this result was not affected by the environmental indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.

Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

Prophage terminase with tRNase activity sensitizes Salmonella enterica to oxidative stress.

Phage viruses shape the evolution and virulence of their bacterial hosts. The Salmonella enterica genome encodes several stress-inducible prophages. The Gifsy-1 prophage terminase protein, whose canonical function is to process phage DNA for packaging in the virus head, unexpectedly acts as a transfer ribonuclease (tRNase) under oxidative stress, cleaving the anticodon loop of tRNALeu. The ensuing RNA fragmentation compromises bacterial translation, intracellular survival, and recovery from oxidative stress in the vertebrate host. S. enterica adapts to this transfer RNA (tRNA) fragmentation by transcribing the RNA repair Rtc system. The counterintuitive translational arrest provided by tRNA cleavage may subvert prophage mobilization and give the host an opportunity for repair as a way of maintaining bacterial genome integrity and ultimately survival in animals.

Biochemical characterization of the meiosis-essential yet evolutionarily divergent topoisomerase VIB-like protein MTOPVIB from Arabidopsis thaliana.

Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have revealed that assembly of a type IIB topoisomerase VI (Topo VI)-like complex, composed of SPO11 and MTOPVIB, is a prerequisite for generating DNA breaks. However, it remains enigmatic if MTOPVIB resembles its Topo VI subunit B (VIB) ortholog in possessing robust ATPase activity, ability to undergo ATP-dependent dimerization, and activation of SPO11-mediated DNA cleavage. Here, we successfully prepared highly pure A. thaliana MTOPVIB and MTOPVIB-SPO11 complex. Contrary to expectations, our findings highlight that MTOPVIB differs from orthologous Topo VIB by lacking ATP-binding activity and independently forming dimers without ATP. Most significantly, our study reveals that while MTOPVIB lacks the capability to stimulate SPO11-mediated DNA cleavage, it functions as a bona fide DNA-binding protein and plays a substantial role in facilitating the dsDNA binding capacity of the MOTOVIB-SPO11 complex. Thus, we illustrate mechanistic divergence between the MTOPVIB-SPO11 complex and classical type IIB topoisomerases.

A Rad50-null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells.

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level.

Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets.

The RNA-guided CRISPR-associated (Cas) enzyme Cas12a cleaves specific double-stranded (ds-) or single-stranded (ss-) DNA targets (in cis), unleashing non-specific ssDNA cleavage (in trans). Though this trans-activity is widely coopted for diagnostics, little is known about target determinants promoting optimal enzyme performance. Using quantitative kinetics, we show formation of activated nuclease proceeds via two steps whereby rapid binding of Cas12a ribonucleoprotein to target is followed by a slower allosteric transition. Activation does not require a canonical protospacer-adjacent motif (PAM), nor is utilization of such PAMs predictive of high trans-activity. We identify several target determinants that can profoundly impact activation times, including bases within the PAM (for ds- but not ssDNA targets) and sequences within and outside those complementary to the spacer, DNA topology, target length, presence of non-specific DNA, and ribose backbone itself, uncovering previously uncharacterized cleavage of and activation by RNA targets. The results provide insight into the mechanism of Cas12a activation, with direct implications on the role of Cas12a in bacterial immunity and for Cas-based diagnostics.

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR-Cas12a.

The CRISPR-Cas12a system has emerged as a powerful tool for next-generation nucleic acid-based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA-enabled trans-cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full-size RNA targets, although LbCas12a exhibits weaker trans-cleavage activity than AsCas12a on both single-stranded DNA and RNA substrates. Remarkably, we find that the RNA-activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA-Sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM-less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double-stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a-based nucleic acid detection and further enhance the understanding of CRISPR-Cas biochemistry.

DNA digestion and formation of DNA-network structures with Holliday junction-resolving enzyme Hjc_15-6 in conjunction with polymerase reactions.

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors.

Mechanism of Viral DNA Packaging in Phage T4 Using Single-Molecule Fluorescence Approaches.

In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring-DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among pac site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms.

Physical interaction with Spo11 mediates the localisation of Mre11 to chromatin in meiosis and promotes its nuclease activity.

Meiotic recombination is of central importance for the proper segregation of homologous chromosomes, but also for creating genetic diversity. It is initiated by the formation of double-strand breaks (DSBs) in DNA catalysed by evolutionarily conserved Spo11, together with additional protein partners. Difficulties in purifying the Spo11 protein have limited the characterization of its biochemical properties and of its interactions with other DSB proteins. In this study, we have purified fragments of Spo11 and show for the first time that Spo11 can physically interact with Mre11 and modulates its DNA binding, bridging, and nuclease activities. The interaction of Mre11 with Spo11 requires its far C-terminal region, which is in line with the severe meiotic phenotypes of various mre11 mutations located at the C-terminus. Moreover, calibrated ChIP for Mre11 shows that Spo11 promotes Mre11 recruitment to chromatin, independent of DSB formation. A mutant deficient in Spo11 interaction severely reduces the association of Mre11 with meiotic chromatin. Consistent with the reduction of Mre11 foci in this mutant, it strongly impedes DSB formation, leading to spore death. Our data provide evidence that physical interaction between Spo11 and Mre11, together with end-bridging, promote normal recruitment of Mre11 to hotspots and DSB formation.

Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding.

The yeast Rif2 protein is known to inhibit Mre11 nuclease and the activation of Tel1 kinase through a short motif termed MIN, which binds the Rad50 subunit and simulates its ATPase activity in vitro. The mechanism by which Rif2 restrains Tel1 activation and the consequences of this inhibition at DNA double-strand breaks (DSBs) are poorly understood. In this study, we employed AlphaFold-Multimer modelling to pinpoint and validate the interaction surface between Rif2 MIN and Rad50. We also engineered the rif2-S6E mutation that enhances the inhibitory effect of Rif2 by increasing Rif2-Rad50 interaction. Unlike rif2Δ, the rif2-S6E mutation impairs hairpin cleavage. Furthermore, it diminishes Tel1 activation by inhibiting Tel1 binding to DSBs while leaving MRX association unchanged, indicating that Rif2 can directly inhibit Tel1 recruitment to DSBs. Additionally, Rif2S6E reduces Tel1-MRX interaction and increases stimulation of ATPase by Rad50, indicating that Rif2 binding to Rad50 induces an ADP-bound MRX conformation that is not suitable for Tel1 binding. The decreased Tel1 recruitment to DSBs in rif2-S6E cells impairs DSB end-tethering and this bridging defect is suppressed by expressing a Tel1 mutant variant that increases Tel1 persistence at DSBs, suggesting a direct role for Tel1 in the bridging of DSB ends.

Biophysical and structural characterization of a multifunctional viral genome packaging motor.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.

MBD4 loss results in global reactivation of promoters and retroelements with low methylated CpG density.

BACKGROUND: Inherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5'-methylcytosine. METHODS: Here, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments. RESULTS: Here we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity. CONCLUSIONS: Our data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions.

Deficiency of macrophage-derived Dnase1L3 causes lupus-like phenotypes in mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by environmental factors and loss of key proteins, including the endonuclease Dnase1L3. Dnase1L3 absence causes pediatric-onset lupus in humans, while reduced activity occurs in adult-onset SLE. The amount of Dnase1L3 that prevents lupus remains unknown. To genetically reduce Dnase1L3 levels, we developed a mouse model lacking Dnase1L3 in macrophages (conditional knockout [cKO]). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Homogeneous and peripheral antinuclear antibodies were detected in the sera by immunofluorescence, consistent with anti-double-stranded DNA (anti-dsDNA) antibodies. Total immunoglobulin M, total immunoglobulin G, and anti-dsDNA antibody levels increased in cKO mice with age. The cKO mice developed anti-Dnase1L3 antibodies. In contrast to global Dnase1L3-/- mice, anti-dsDNA antibodies were not elevated early in life. The cKO mice had minimal kidney pathology. Therefore, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes, and macrophage-derived DnaselL3 helps limit lupus.

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.