ABSTRACT
The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements.
Subject(s)
Adenosine Diphosphate , Adenosine Triphosphate , Cryoelectron Microscopy , DNA Transposable Elements , Transposases , DNA Transposable Elements/genetics , Adenosine Triphosphate/metabolism , Transposases/metabolism , Transposases/genetics , Transposases/chemistry , Adenosine Diphosphate/metabolism , Protein Binding , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Models, Molecular , Protein Multimerization , Binding SitesABSTRACT
BRCC36 is a Zn(2+)-dependent deubiquitinating enzyme (DUB) that hydrolyzes lysine-63-linked ubiquitin chains as part of distinct macromolecular complexes that participate in either interferon signaling or DNA-damage recognition. The MPN(+) domain protein BRCC36 associates with pseudo DUB MPN(-) proteins KIAA0157 or Abraxas, which are essential for BRCC36 enzymatic activity. To understand the basis for BRCC36 regulation, we have solved the structure of an active BRCC36-KIAA0157 heterodimer and an inactive BRCC36 homodimer. Structural and functional characterizations show how BRCC36 is switched to an active conformation by contacts with KIAA0157. Higher-order association of BRCC36 and KIAA0157 into a dimer of heterodimers (super dimers) was required for DUB activity and interaction with targeting proteins SHMT2 and RAP80. These data provide an explanation of how an inactive pseudo DUB allosterically activates a cognate DUB partner and implicates super dimerization as a new regulatory mechanism underlying BRCC36 DUB activity, subcellular localization, and biological function.
Subject(s)
Ants/enzymology , Insect Proteins/chemistry , Nuclear Matrix-Associated Proteins/chemistry , Ubiquitin-Specific Proteases/chemistry , Animals , Catalytic Domain , Crystallography, X-Ray , Deubiquitinating Enzymes , HEK293 Cells , HeLa Cells , Humans , Insect Proteins/physiology , Kinetics , Membrane Proteins/chemistry , Models, Molecular , Nuclear Matrix-Associated Proteins/physiology , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Secondary , Ubiquitin-Specific Proteases/physiologyABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , Crossing Over, Genetic , Exodeoxyribonucleases/metabolism , Meiosis/genetics , MutL Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Cell Cycle Proteins/genetics , Chromosomes, Fungal , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Models, Biological , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Recombination, GeneticABSTRACT
DNA nanotubes (NTs) have attracted extensive interest as artificial cytoskeletons for biomedical, synthetic biology, and materials applications. Here, we report the modular design and assembly of a minimalist yet robust DNA wireframe nanotube with tunable cross-sectional geometry, cavity size, chirality, and length, while using only four DNA strands. We introduce an h-motif structure incorporating double-crossover (DX) tile-like DNA edges to achieve structural rigidity and provide efficient self-assembly of h-motif-based DNA nanotube (H-NT) units, thus producing programmable, micrometer-long nanotubes. We demonstrate control of the H-NT nanotube length via short DNA modulators. Finally, we use an enzyme, RNase H, to take these structures out of equilibrium and trigger nanotube assembly at a physiologically relevant temperature, underlining future cellular applications. The minimalist H-NTs can assemble at near-physiological salt conditions and will serve as an easily synthesized, DNA-economical modular template for biosensors, plasmonics, or other functional materials and as cost-efficient drug-delivery vehicles for biomedical applications.
Subject(s)
Biosensing Techniques , Nanotubes , Nanotechnology , Nanotubes/chemistry , DNA/chemistry , DNA ReplicationABSTRACT
RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.
Subject(s)
Adenine Nucleotides/chemistry , Endoribonucleases/chemistry , Oligoribonucleotides/chemistry , Adenosine Diphosphate/chemistry , Adenylyl Imidodiphosphate/chemistry , Animals , Ankyrin Repeat , Binding Sites , Crystallography, X-Ray , Dimerization , Encephalomyocarditis virus , Endoribonucleases/genetics , Endoribonucleases/physiology , HeLa Cells , Humans , Models, Molecular , Mutagenesis, Site-Directed , Picornaviridae , Protein Structure, Tertiary , Scattering, Radiation , Structure-Activity Relationship , Sus scrofaABSTRACT
Zika virus (ZIKV), a member of the Flaviviridae family, has emerged as a major public health threat, since ZIKV infection has been connected to microcephaly and other neurological disorders. Flavivirus genome replication is driven by NS5, an RNA-dependent RNA polymerase (RdRP) that also contains a N-terminal methyltransferase domain essential for viral mRNA capping. Given its crucial roles, ZIKV NS5 has become an attractive antiviral target. Here, we have used integrated structural biology approaches to characterize the supramolecular arrangement of the full-length ZIKV NS5, highlighting the assembly and interfaces between NS5 monomers within a dimeric structure, as well as the dimer-dimer interactions to form higher order fibril-like structures. The relative orientation of each monomer within the dimer provides a model to explain the coordination between MTase and RdRP domains across neighboring NS5 molecules and mutational studies underscore the crucial role of the MTase residues Y25, K28 and K29 in NS5 dimerization. The basic residue K28 also participates in GTP binding and competition experiments indicate that NS5 dimerization is disrupted at high GTP concentrations. This competition represents a first glimpse at a molecular level explaining how dimerization might regulate the capping process.
Subject(s)
Protein Conformation , Protein Multimerization , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Zika Virus/enzymology , Crystallography, X-Ray , Humans , Models, Molecular , Protein Binding , RNA-Dependent RNA Polymerase/metabolism , Viral Nonstructural Proteins/metabolismABSTRACT
Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.
Subject(s)
GTP Phosphohydrolases/chemistry , RNA, Ribosomal/chemistry , Ribosomal Proteins/chemistry , Bacillus subtilis/chemistry , Bacillus subtilis/genetics , Cryoelectron Microscopy , GTP Phosphohydrolases/ultrastructure , Hydrolysis , Models, Molecular , Protein Conformation , RNA, Ribosomal/genetics , RNA, Ribosomal/ultrastructure , Ribosomal Proteins/ultrastructure , Ribosome Subunits, Large, Bacterial/chemistry , Ribosome Subunits, Large, Bacterial/genetics , Ribosome Subunits, Large, Bacterial/ultrastructure , Ribosomes/genetics , Ribosomes/ultrastructureABSTRACT
The ß-clamp is a protein hub central to DNA replication and fork management. Proteins interacting with the ß-clamp harbor a conserved clamp-binding motif that is often found in extended regions. Therefore, clamp interactions have -almost exclusively- been studied using short peptides recapitulating the binding motif. This approach has revealed the molecular determinants that mediate the binding but cannot describe how proteins with clamp-binding motifs embedded in structured domains are recognized. The mismatch repair protein MutL has an internal clamp-binding motif, but its interaction with the ß-clamp has different roles depending on the organism. In Bacillus subtilis, the interaction stimulates the endonuclease activity of MutL and it is critical for DNA mismatch repair. Conversely, disrupting the interaction between Escherichia coli MutL and the ß-clamp only causes a mild mutator phenotype. Here, we determined the structures of the regulatory domains of E. coli and B. subtilis MutL bound to their respective ß-clamps. The structures reveal different binding modes consistent with the binding to the ß-clamp being a two-step process. Functional characterization indicates that, within the regulatory domain, only the clamp binding motif is required for the interaction between the two proteins. However, additional motifs beyond the regulatory domain may stabilize the interaction. We propose a model for the activation of the endonuclease activity of MutL in organisms lacking methyl-directed mismatch repair.
Subject(s)
DNA Polymerase III/genetics , DNA Replication/genetics , Escherichia coli Proteins/genetics , MutL Proteins/genetics , Adenosine Triphosphatases , Bacillus subtilis/chemistry , Bacillus subtilis/genetics , Binding Sites/genetics , DNA Mismatch Repair/genetics , DNA Polymerase III/chemistry , Escherichia coli/genetics , Models, Molecular , MutL Proteins/chemistry , Protein Binding , Species SpecificityABSTRACT
Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.
Subject(s)
Escherichia coli Proteins/metabolism , GTP-Binding Proteins/metabolism , Homeostasis , RNA, Ribosomal, 16S/metabolism , RNA-Binding Proteins/metabolism , Ribosome Subunits, Small, Bacterial/metabolism , Ribosome Subunits, Small/metabolism , Base Sequence , Binding Sites , Cryoelectron Microscopy , Escherichia coli Proteins/genetics , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/genetics , Nucleic Acid Conformation , Protein Binding , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , RNA-Binding Proteins/genetics , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosome Subunits, Small/genetics , Ribosome Subunits, Small/ultrastructure , Ribosome Subunits, Small, Bacterial/genetics , Ribosome Subunits, Small, Bacterial/ultrastructureABSTRACT
Recent work suggests that bacterial YjeQ (RsgA) participates in the late stages of assembly of the 30S subunit and aids the assembly of the decoding center but also binds the mature 30S subunit with high affinity. To determine the function and mechanisms of YjeQ in the context of the mature subunit, we determined the cryo-EM structure of the fully assembled 30S subunit in complex with YjeQ at 5.8-Å resolution. We found that binding of YjeQ stabilizes helix 44 into a conformation similar to that adopted by the subunit during proofreading. This finding indicates that, along with acting as an assembly factor, YjeQ has a role as a checkpoint protein, consisting of testing the proofreading ability of the 30S subunit. The structure also informs the mechanism by which YjeQ implements the release from the 30S subunit of a second assembly factor, called RbfA. Finally, it reveals how the 30S subunit stimulates YjeQ GTPase activity and leads to release of the protein. Checkpoint functions have been described for eukaryotic ribosome assembly factors; however, this work describes an example of a bacterial assembly factor that tests a specific translation mechanism of the 30S subunit.
Subject(s)
Cryoelectron Microscopy , Escherichia coli K12/chemistry , Escherichia coli Proteins/chemistry , GTP Phosphohydrolases/chemistry , Ribosome Subunits, Small, Bacterial/chemistry , Ribosome Subunits, Small, Bacterial/ultrastructure , Escherichia coli K12/metabolism , Escherichia coli K12/ultrastructure , Escherichia coli Proteins/metabolism , GTP Phosphohydrolases/metabolism , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosome Subunits, Small, Bacterial/metabolismABSTRACT
Nucleic acids are potential pathogen-associated or danger-associated molecular patterns that modulate immune responses and the development of autoimmune disorders. Class A scavenger receptors (SR-As) are a diverse group of pattern recognition receptors that recognize a variety of polyanionic ligands including nucleic acids. While SR-As are important for the recognition and internalization of extracellular dsRNA, little is known about extracellular DNA, despite its association with chronic infections and autoimmune disorders. In this study, we investigated the specificity of and requirement for SR-As in binding and internalizing different species, sequences and lengths of nucleic acids. We purified recombinant coiled-coil/collagenous and scavenger receptor cysteine-rich (SRCR) domains that have been implicated as potential ligand-binding domains. We detected a direct interaction of RNA and DNA species with the coiled-coil/collagenous domain, but not the SRCR domain. Despite the presence of additional surface receptors that bind nucleic acids, SR-As were found to be sufficient for nucleic acid binding and uptake in A549 human lung epithelial cells. Moreover, these findings suggest that the coiled-coil/collagenous domain of SR-As is sufficient to bind nucleic acids independent of species, sequence or length.
Subject(s)
Nucleic Acids/metabolism , RNA, Double-Stranded/metabolism , Scavenger Receptors, Class A/metabolism , Virus Internalization , A549 Cells , Amino Acid Sequence , Humans , Nucleic Acids/immunology , Receptors, Pattern Recognition , Scavenger Receptors, Class A/immunologyABSTRACT
DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn(2+)-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.
Subject(s)
Adenosine Triphosphatases/chemistry , Bacillus subtilis/enzymology , Adenosine Triphosphatases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Conserved Sequence , Crystallography, X-Ray , DNA Mismatch Repair , Endonucleases/chemistry , Enzyme Activation , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Zinc/metabolismABSTRACT
KEOPS is an ancient protein complex required for the biosynthesis of N6-threonylcarbamoyladenosine (t(6)A), a universally conserved tRNA modification found on all ANN-codon recognizing tRNAs. KEOPS consist minimally of four essential subunits, namely the proteins Kae1, Bud32, Cgi121 and Pcc1, with yeast possessing the fifth essential subunit Gon7. Bud32, Cgi121, Pcc1 and Gon7 appear to have evolved to regulate the central t(6)A biosynthesis function of Kae1, but their precise function and mechanism of action remains unclear. Pcc1, in particular, binds directly to Kae1 and by virtue of its ability to form dimers in solution and in crystals, Pcc1 was inferred to function as a dimerization module for Kae1 and therefore KEOPS. We now present a 3.4 Å crystal structure of a dimeric Kae1-Pcc1 complex providing direct evidence that Pcc1 can bind and dimerize Kae1. Further biophysical analysis of a complete archaeal KEOPS complex reveals that Pcc1 facilitates KEOPS dimerization in vitro Interestingly, while Pcc1-mediated dimerization of KEOPS is required to support the growth of yeast, it is dispensable for t(6)A biosynthesis by archaeal KEOPS in vitro, raising the question of how precisely Pcc1-mediated dimerization impacts cellular biology.
Subject(s)
Adenosine/analogs & derivatives , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Multiprotein Complexes/metabolism , Protein Multimerization , Pyrococcus furiosus/metabolism , Adenosine/biosynthesis , Biophysical Phenomena , Chromatography, Gel , Crystallography, X-Ray , Scattering, Radiation , Scattering, Small Angle , Solutions , Structure-Activity Relationship , X-Ray DiffractionABSTRACT
Most proteins function within networks and, therefore, protein interactions are central to protein function. Although stable macromolecular machines have been extensively studied, dynamic protein interactions remain poorly understood. Small-angle X-ray scattering probes the size, shape and dynamics of proteins in solution at low resolution and can be used to study samples in a large range of molecular weights. Therefore, it has emerged as a powerful technique to study the structure and dynamics of biomolecular systems and bridge fragmented information obtained using high-resolution techniques. Here we review how small-angle X-ray scattering can be combined with other structural biology techniques to study protein dynamics. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.
Subject(s)
Crystallography, X-Ray/methods , Proteins/chemistry , Crystallography, X-Ray/instrumentation , Protein Domains , Scattering, Small AngleABSTRACT
Saccharomyces cerevisiae Saw1 is an essential gene in single-strand annealing - the DNA repair pathway that repairs double-strand breaks when they occur between homologous repeats. Saw1 interacts with the structure-specific nuclease Rad1-Rad10 and this results in the recruitment of this nuclease to 3' non-homologous DNA tailed recombination intermediates. Saw1 is unstable in the absence of the Rad1-Rad10 nuclease and, hence, it has been difficult to study its specific function in vitro. In the present work, we present the combination of dynamic light scattering and differential scanning fluorimetry techniques to optimize the stability and homogeneity of recombinant Saw1. The protein expression and purification conditions identified in this study allow for higher recovery of soluble Saw1 and enable the biochemical characterization of the protein.
Subject(s)
DNA-Binding Proteins , Escherichia coli/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Escherichia coli/chemistry , Escherichia coli/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purificationABSTRACT
The bacterial transposon Tn7 facilitates horizontal transfer by directing transposition into actively replicating DNA with the element-encoded protein TnsE. Structural analysis of the C-terminal domain of wild-type TnsE identified a novel protein fold including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-activity variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structure-based analysis of a series of TnsE mutants relates transposition activity to DNA binding stability. Wild-type TnsE appears to naturally form an unstable complex with a target DNA, whereas mutant combinations required for large changes in transposition frequency and targeting stabilized this interaction. Collectively, our work unveils a unique structural proofreading mechanism where toggling between two conformations regulates target commitment by limiting the stability of target DNA engagement until an appropriate insertion site is identified.
Subject(s)
Bacterial Proteins/chemistry , DNA Transposable Elements , DNA-Binding Proteins/chemistry , Transposases/metabolism , Alanine/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Models, Molecular , Mutation , Protein Folding , Protein Structure, TertiaryABSTRACT
The sliding clamp enhances polymerase processivity and coordinates DNA replication with other critical DNA processing events including translesion synthesis, Okazaki fragment maturation and DNA repair. The relative binding affinity of the sliding clamp for its partners determines how these processes are orchestrated and is essential to ensure the correct processing of newly replicated DNA. However, while stable clamp interactions have been extensively studied; dynamic interactions mediated by the sliding clamp remain poorly understood. Here, we characterize the interaction between the bacterial sliding clamp (ß-clamp) and one of its weak-binding partners, the DNA mismatch repair protein MutL. Disruption of this interaction causes a mild mutator phenotype in Escherichia coli, but completely abrogates mismatch repair activity in Bacillus subtilis. We stabilize the MutL-ß interaction by engineering two cysteine residues at variable positions of the interface. Using disulfide bridge crosslinking, we have stabilized the E. coli and B. subtilis MutL-ß complexes and have characterized their structures using small angle X-ray scattering. We find that the MutL-ß interaction greatly stimulates the endonuclease activity of B. subtilis MutL and supports this activity even in the absence of the N-terminal region of the protein.
Subject(s)
Adenosine Triphosphatases/chemistry , Bacterial Proteins/chemistry , DNA Polymerase III/chemistry , Endodeoxyribonucleases/chemistry , Escherichia coli Proteins/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cysteine/genetics , DNA/metabolism , DNA Polymerase III/genetics , DNA Polymerase III/metabolism , Endodeoxyribonucleases/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Models, Molecular , MutL Proteins , Protein Binding , Protein Structure, TertiaryABSTRACT
Macrophage receptor with collagenous structure (MARCO) is a class A scavenger receptor (cA-SR) that recognizes and phagocytoses a wide variety of pathogens. Most cA-SRs that contain a C-terminal scavenger receptor cysteine-rich (SRCR) domain use the proximal collagenous domain to bind ligands. In contrast, the role of the SRCR domain of MARCO in phagocytosis, adhesion and pro-inflammatory signaling is less clear. The discovery of a naturally occurring transcript variant lacking the SRCR domain, MARCOII, provided the opportunity to study the role of the SRCR domain of MARCO. We tested whether the SRCR domain is required for ligand binding, promoting downstream signaling and enhancing cellular adhesion. Unlike cells expressing full-length MARCO, ligand binding was abolished in MARCOII-expressing cells. Furthermore, co-expression of MARCO and MARCOII impaired phagocytic function, indicating that MARCOII acts as a dominant-negative variant. Unlike MARCO, expression of MARCOII did not enhance Toll-like receptor 2 (TLR2)-mediated pro-inflammatory signaling in response to bacterial stimulation. MARCO-expressing cells were more adherent and exhibited a dendritic-like phenotype, whereas MARCOII-expressing cells were less adherent and did not exhibit changes in morphology. These data suggest the SRCR domain of MARCO is the key domain in modulating ligand binding, enhancing downstream pro-inflammatory signaling and MARCO-mediated cellular adhesion.
Subject(s)
Alternative Splicing/genetics , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Amino Acid Sequence , Animals , Cell Adhesion , Cell Shape , Cloning, Molecular , Endocytosis , HEK293 Cells , Humans , Ligands , Lipopolysaccharide Receptors/metabolism , Mice, Inbred C57BL , NF-kappa B/metabolism , Protein Domains , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Immunologic/metabolism , Streptococcus pneumoniae/physiology , Structure-Activity Relationship , Toll-Like Receptor 2/metabolismABSTRACT
Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA, Cruciform/metabolism , DNA, Fungal/metabolism , DNA-Binding Proteins/metabolism , Deoxyribonuclease I/metabolism , Meiosis/physiology , MutS Homolog 2 Protein/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adaptor Proteins, Signal Transducing/genetics , DNA Breaks, Single-Stranded , DNA, Cruciform/genetics , DNA, Fungal/genetics , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , DNA-Binding Proteins/genetics , Deoxyribonuclease I/genetics , MutL Protein Homolog 1 , MutL Proteins , MutS Homolog 2 Protein/genetics , MutS Homolog 3 Protein , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules.