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In bacterial cells, processing of double‐stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence Chi and is catalysed by either an AddAB‐ or RecBCD‐type helicase–nuclease. Here, we report the crystal structure of AddAB bound to DNA. The structure allows identification of a putative Chi‐recognition site in an inactivated helicase domain of the AddB subunit. By generating mutant protein complexes that do not respond to Chi, we show that residues responsible for Chi recognition are located in positions equivalent to the signature motifs of a conventional helicase. Comparison with the related RecBCD complex, which recognizes a different Chi sequence, provides further insight into the structural basis for sequence‐specific ssDNA recognition. The structure suggests a simple mechanism for DNA break processing, explains how AddAB and RecBCD can accomplish the same overall reaction with different sets of functional modules and reveals details of the role of an Fe–S cluster in protein stability and DNA binding. Homologous recombination DNA repair requires double‐strand break resection by helicase–nuclease enzymes. The crystal structure of bacterial AddAB in complex with DNA substrates shows that it employs an inactive helicase site to recognize ‘Chi’ recombination hotspot sequences that regulate resection.

BRCA2, the breast cancer susceptibility gene factor, interacts with TREX-2, a protein complex involved in the biogenesis and export of messenger ribonucleoprotein, to process DNA–RNA hybrid structures called R-loops that can trigger genome instability; these may be a central cause of the stress occurring in early cancer cells that drives oncogenesis. Harnessing an R-loop to promote cancer R-loops — naturally occurring three-stranded nucleic acid structures consisting of an RNA–DNA hybrid and displaced single-stranded DNA — are among the potential inducers of genome instability. This study shows that TREX-2, a complex involved in the biogenesis and export of messenger ribonucleoprotein (mRNP), interacts with the breast cancer susceptibility gene factor BRCA2 to process R-loops. Human cells depleted of BRCA2 accumulate high levels of R-loops. This unexpected interaction between tumour suppressors and R-loops suggests that R-loops may be a major cause of replication stress and tumorigenicity. Genome instability is central to ageing, cancer and other diseases. It is not only proteins involved in DNA replication or the DNA damage response (DDR) that are important for maintaining genome integrity: from yeast to higher eukaryotes, mutations in genes involved in pre-mRNA splicing and in the biogenesis and export of messenger ribonucleoprotein (mRNP) also induce DNA damage and genome instability. This instability is frequently mediated by R-loops formed by DNA–RNA hybrids and a displaced single-stranded DNA 1 . Here we show that the human TREX-2 complex, which is involved in mRNP biogenesis and export, prevents genome instability as determined by the accumulation of γ-H2AX (Ser-139 phosphorylated histone H2AX) and 53BP1 foci and single-cell electrophoresis in cells depleted of the TREX-2 subunits PCID2, GANP and DSS1. We show that the BRCA2 repair factor, which binds to DSS1, also associates with PCID2 in the cell. The use of an enhanced green fluorescent protein-tagged hybrid-binding domain of RNase H1 and the S9.6 antibody did not detect R-loops in TREX-2-depleted cells, but did detect the accumulation of R-loops in BRCA2-depleted cells. The results indicate that R-loops are frequently formed in cells and that BRCA2 is required for their processing. This link between BRCA2 and RNA-mediated genome instability indicates that R-loops may be a chief source of replication stress and cancer-associated instability.

FAN1 is a DNA dependent nuclease whose proper function is essential for maintaining human health. For example, a genetic variant in FAN1, Arg507 to His hastens onset of Huntington’s disease, a repeat expansion disorder for which there is no cure. How the Arg507His mutation affects FAN1 structure and enzymatic function is unknown. Using cryo-EM and biochemistry, we have discovered that FAN1 arginine 507 is critical for its interaction with PCNA, and mutation of Arg507 to His attenuates assembly of the FAN1–PCNA complex on a disease-relevant extrahelical DNA extrusions formed within DNA repeats. This mutation concomitantly abolishes PCNA–FAN1–dependent cleavage of such extrusions, thus unraveling the molecular basis for a specific mutation in FAN1 that dramatically hastens the onset of Huntington’s disease. These results underscore the importance of PCNA to the genome stabilizing function of FAN1. FAN1 nuclease removes DNA triplet repeat loops by a process that requires PCNA. Using cryo-EM, the authors elucidate this mechanism, and show that a Huntington’s disease modifying R507H mutation inactivates FAN1 by compromising the FAN1-PCNA complex.

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.

Cells dedicate significant energy to build proteins often organized in multiprotein assemblies with tightly regulated stoichiometries. As genes encoding subunits assembling in a multisubunit complex are dispersed in the genome of eukaryotes, it is unclear how these protein complexes assemble. Here, we show that mammalian nuclear transcription complexes (TFIID, TREX-2 and SAGA) composed of a large number of subunits, but lacking precise architectural details are built co-translationally. We demonstrate that dimerization domains and their positions in the interacting subunits determine the co-translational assembly pathway (simultaneous or sequential). The lack of co-translational interaction can lead to degradation of the partner protein. Thus, protein synthesis and complex assembly are linked in building mammalian multisubunit complexes, suggesting that co-translational assembly is a general principle in mammalian cells to avoid non-specific interactions and protein aggregation. These findings will also advance structural biology by defining endogenous co-translational building blocks in the architecture of multisubunit complexes. Genes encoding protein complex subunits are often dispersed in the genome of eukaryotes, raising the question how these protein complexes assemble. Here, the authors provide evidence that mammalian nuclear transcription complexes are formed co-translationally to ensure specific and functional interactions.

The endonuclease activity of MLH1-MLH3 (MutLγ) is stimulated by MSH4-MSH5 (MutSγ), EXO1, and RFC-PCNA to resolve meiotic recombination intermediates such as double Holliday junctions (HJs) into crossovers. We show that EXO1 directly interacts with MLH1 via the EXO1 MIP motif and a patch centered around EXO1-I403. Disrupting this interaction unexpectedly only partially inhibited MutLγ. We found that EXO1 also directly interacts with MutSγ. Crucially, a single point mutation in EXO1 (W371E) impairs its interaction with MSH4 and completely abolished its ability to activate DNA nicking by MutLγ without affecting its intrinsic nuclease function. Finally, disrupting magnesium coordinating residues in the nuclease domain of EXO1 has no impact on MutSγ-MutLγ activity, while the integrity of EXO1 residues mediating interactions with double-stranded DNA (dsDNA) is important. Our findings suggest EXO1 is an integral structural component of the meiotic resolvase complex, supported by conserved interactions with MutSγ, MutLγ and dsDNA. We propose that EXO1 helps tether MutSγ-MutLγ to dsDNA downstream of HJ recognition to promote DNA cleavage. EXO1 has a structural role to promote the MLH1-MLH3 nuclease complex to process meiotic recombination intermediates into crossovers. Here, the authors demonstrate that EXO1 function depends on conserved interactions with the MSH4 subunit of MSH4-MSH5, the MLH1 subunit of MLH1-MLH3, and DNA.

The nuclear export of mRNA represents a critical regulatory node in eukaryotic gene expression. This process is orchestrated by two conserved multi-subunit assemblies: the transcription-and-export complex (TREX) and TREX-2. While TREX facilitates mRNP packaging through multivalent RNA-protein interactions, the precise mechanism by which TREX-2 contributes to mRNA export has remained elusive. Here, we report a functional interaction between UAP56 and TREX-2 and resolve the structures of TREX-2 in both apo and UAP56-bound states. UAP56 engages TREX-2 via its N-terminal region, positioning its RecA domains on the V-shaped surface of the complex. A conserved loop from TREX-2 inserts between the RecA domains of UAP56, stabilizing an open conformation. Biochemical assays demonstrate that TREX-2 significantly stimulates the ATPase activity of UAP56, thereby promoting RNA release. These findings provide structural and mechanistic insights into TREX-2-mediated regulation of mRNA export through UAP56 remodeling. The nuclear export of mRNA is a crucial regulatory step in eukaryotic gene expression. Here, the authors show that TREX-2 interacts with UAP56, enhances its ATPase activity, and promotes mRNA release from UAP56. These findings provide key insights into the mechanisms underlying mRNA export.

The TREX1 gene encodes a potent DNA exonuclease, and mutations in TREX1 cause a spectrum of lupus-like autoimmune diseases. Most lupus patients develop autoantibodies to double-stranded DNA (dsDNA), but the source of DNA antigen is unknown. The TREX1 D18N mutation causes a monogenic, cutaneous form of lupus called familial chilblain lupus, and the TREX1 D18N enzyme exhibits dysfunctional dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune disease. We determined the structure of the TREX1 D18N protein in complex with dsDNA, revealing how this exonuclease uses a novel DNA-unwinding mechanism to separate the polynucleotide strands for single-stranded DNA (ssDNA) loading into the active site. The TREX1 D18N dsDNA interactions coupled with catalytic deficiency explain how this mutant nuclease prevents dsDNA degradation. We tested the effects of TREX1 D18N in vivo by replacing the TREX1 WT gene in mice with the TREX1 D18N allele. The TREX1 D18N mice exhibit systemic inflammation, lymphoid hyperplasia, vasculitis, and kidney disease. The observed lupus-like inflammatory disease is associated with immune activation, production of autoantibodies to dsDNA, and deposition of immune complexes in the kidney. Thus, dysfunctional dsDNA degradation by TREX1 D18N induces disease in mice that recapitulates many characteristics of human lupus. Failure to clear DNA has long been linked to lupus in humans, and these data point to dsDNA as a key substrate for TREX1 and a major antigen source in mice with dysfunctional TREX1 enzyme. Significance The TREX1 enzyme degrades DNA, and mutations in the TREX1 gene cause autoimmune diseases. The TREX1 D18N mutation causes a form of lupus called familial chilblain lupus. We solved the structure of TREX1 D18N bound to dsDNA, showing how the enzyme interacts with dsDNA. We also replaced the TREX1 WT gene in mice with the TREX1 D18N mutated gene and showed how this mutation causes a lupus-like disease. Together, the TREX1 D18N–dsDNA structure and the spontaneous disease exhibited in the TREX1 D18N mouse help to define how TREX1 degrades dsDNA to prevent this molecule from acting as an autoantigen in the mouse and, most likely, in humans to promote autoimmune disease.

The Cas4 protein is one of the core CRISPR-associated (Cas) proteins implicated in the prokaryotic CRISPR system for antiviral defence. Cas4 is thought to play a role in the capture of new viral DNA sequences for incorporation into the host genome. No biochemical activity has been reported for Cas4, but it is predicted to include a RecB nuclease domain. We show here that Cas4 family proteins from the archaeon Sulfolobus solfataricus utilise four conserved cysteine residues to bind an iron-sulfur cluster in an arrangement reminiscent of the AddB nuclease of Bacillus subtilis. The Cas4 family protein Sso0001 is a 5' to 3' single stranded DNA exonuclease in vitro that is stalled by extrahelical DNA adducts. A role for Cas4 in DNA duplex strand resectioning to generate recombinogenic 3' single stranded DNA overhangs is proposed. Comparison of the AddB structure with that of a related bacterial nuclease from Eubacterium rectales reveals that the iron-sulfur cluster can be replaced by a zinc ion without disrupting the protein structure, with implications for the evolution of iron-sulfur binding proteins.

Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes, such as aneuploidy. The Mlh1–Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and polo kinase Cdc5. Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ–Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, noncatalytic role for Exo1, through noncanonical interaction with polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species.