SUMMARY Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of novel sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUSs) uncovers which PUSs modify each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided PUSs. Upon heat shock in yeast, Pus7-mediated pseudouridylation is induced at >200 sites and Pus7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved, but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcritome-wide scope for pseudouridine, and methods to dissect its underlying mechanisms and function.

In yeast, the impact of gene knockouts depends on genetic background.

RecQ family helicases catalyze critical genome maintenance reactions in bacterial and eukaryotic cells, playing key roles in several DNA metabolic processes. Mutations in recQ genes are linked to genome instability and human disease. To define the physical basis of RecQ enzyme function, we have determined a 1.8 Å resolution crystal structure of the catalytic core of Escherichia coli RecQ in its unbound form and a 2.5 Å resolution structure of the core bound to the ATP analog ATPγS. The RecQ core comprises four conserved subdomains; two of these combine to form its helicase region, while the others form unexpected Zn2+‐binding and winged‐helix motifs. The structures reveal the molecular basis of missense mutations that cause Bloom's syndrome, a human RecQ‐associated disease. Finally, based on findings from the structures, we propose a mechanism for RecQ activity that could explain its functional coordination with topoisomerase III.

Bloom's syndrome is a hereditary cancer-predisposition disorder resulting from mutations in the BLM gene. In humans, BLM encodes one of five members of the RecQ helicase family. One function of BLM is to act in concert with topoisomerase IIIa (TOPO IIIa) to resolve recombination intermediates containing double Holliday junctions by a process called double Holliday junction dissolution, herein termed dissolution. Here, we show that dissolution is highly specific for BLM among human RecQ helicases and critically depends upon a functional HRDC domain in BLM. We show that the HRDC domain confers DNA structure specificity, and is required for the efficient binding to and unwinding of double Holliday junctions, but not for the unwinding of a simple partial duplex substrate. Furthermore, we show that lysine-1270 of BLM, which resides in the HRDC domain and is predicted to play a role in mediating interactions with DNA, is required for efficient dissolution.

RecQ DNA helicases are critical components of DNA replication, recombination, and repair machinery in all eukaryotes and bacteria. Eukaryotic RecQ helicases are known to associate with numerous genome maintenance proteins that modulate their cellular functions, but there is little information regarding protein complexes involving the prototypical bacterial RecQ proteins. Here we use an affinity purification scheme to identify three heterologous proteins that associate with Escherichia coli RecQ: SSB (single-stranded DNA-binding protein), exonuclease I, and RecJ exonuclease. The RecQ-SSB interaction is direct and is mediated by the RecQ winged helix subdomain and the C terminus of SSB. Interaction with SSB has important functional consequences for RecQ. SSB stimulates RecQ-mediated DNA unwinding, whereas deletion of the C-terminal RecQ-binding site from SSB produces a variant that blocks RecQ DNA binding and unwinding activities, suggesting that RecQ recognizes both the SSB C terminus and DNA in SSB⅐DNA nucleoprotein complexes. These findings, together with the noted interactions between human RecQ proteins and Replication Protein A, identify SSB as a broadly conserved RecQ-binding protein. These results also provide a simple model that explains RecQ integration into genome maintenance processes in E. coli through its association with SSB.

Thrombospondins (TSPs) are secreted glycoproteins that play key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6 Å resolution crystal structure of the glycosylated signature domain of human TSP-2, which includes three epidermal growth factor-like (EGF-like) modules, 13 aspartate-rich repeats, and a lectin-like module. These elements interact extensively to form three striking structural regions termed the stalk, wire, and globe. The TSP-2 signature domain is stabilized by these interactions and by a network of 30 bound Ca 2+ ions and 18 disulfide bonds. The structure suggests how genetic alterations of TSPs result in disease.Keywords calcium binding; extracellular matrix; lectin-like module; skeletal dysplasia; epidermal growth factor-like module; protein crystallography Thrombospondins (TSPs) are a family of five secreted multimodular metallo/glycoproteins that have diverse roles involving interactions between cells and the extracellular matrix {Adams, 2004 #3568}. For TSP-2, these functions are critical for such processes as synaptogenesis{Christopherson, 2005 #3578}, megakaryocytopoiesis{Kyriakides, 2003, and the foreign body reaction{Kyriakides, 2001 #3588}. All TSPs contain a highly conserved "signature domain" consisting of tandem epidermal growth factor-like (EGF-like) modules, aspartate-rich repeats, and a lectin-like module at their C-termini ( Fig. 1a). These modules are conserved with remarkable fidelity as is evidenced by a 458-residue stretch of fly TSP and human TSP-2 in the signature domain that are 60% identical without an insertion or deletion{Adams, 2003 #3571;LaBell, 1993 #340}. The signature domain contains the sites of polymorphisms and mutations linked to familial coronary artery disease and two skeletal disorders, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1){Topol, 2001 #3537; Kennedy, 2005 #3585;Posey, 2004 #3579}. Here, we describe the 2.6 Å resolution crystal structure of the complete glycosylated signature domain of human TSP-2, which includes three epidermal growth factor-like (EGF-like) modules, 13 aspartate-rich repeats, and a lectin-like module, along with 30 bound Ca 2+ ions. The structure reveals the highly intertwined nature of the signature domain that includes major interactions among the different parts of the molecule. The structure furthermore establishes a molecular explanation of how known genetic alterations of TSPs result in disease.Correspondence and requests for materials should be addressed to D. NIH Public Access Results Structure of the human TSP-2 signature domainThe crystal structure of the signature domain of human TSP-2 was solved using a combination of anomalous scattering and molecular replacement approaches. Electron-density maps, particularly those derived from molecular replacement using the structure of a fragment of the signature domain from TSP-1{Kvansakul, 2004 #3561}, were of high quality ( Fig. 1b), and allowed modelling of the entire domain structure.The overall structu...

RecQ DNA helicases are multidomain enzymes that play pivotal roles in genome maintenance pathways. While the ATPase and helicase activities of these enzymes can be attributed to the conserved catalytic core domain, the role of the Helicase-and-RNase-D-C-terminal (HRDC) domain in RecQ function has yet to be elucidated. Here, we report the crystal structure of the E. coli RecQ HRDC domain, revealing a globular fold that resembles known DNA binding domains. We show that this domain preferentially binds single-stranded DNA and identify its DNA binding surface. HRDC domain mutations in full-length RecQ lead to surprising differences in its structure-specific DNA binding properties. These data support a model in which naturally occurring variations in DNA binding residues among diverse RecQ homologs serve to target these enzymes to distinct substrates and provide insight into a mechanism whereby RecQ enzymes have evolved distinct functions in organisms that encode multiple recQ genes.

Single-stranded DNA (ssDNA)-binding (SSB) proteins are uniformly required to bind and protect single-stranded intermediates in DNA metabolic pathways. All bacterial and eukaryotic SSB proteins studied to date oligomerize to assemble four copies of a conserved domain, called an oligonucleotide͞oligosaccharide-binding (OB) fold, that cooperate in nonspecific ssDNA binding. The vast majority of bacterial SSB family members function as homotetramers, with each monomer contributing a single OB fold. However, SSB proteins from the Deinococcus-Thermus genera are exceptions to this rule, because they contain two OB folds per monomer. To investigate the structural consequences of this unusual arrangement, we have determined a 1.8-Å-resolution x-ray structure of Deinococcus radiodurans SSB. The structure shows that D. radiodurans SSB comprises two OB domains linked by a ␤-hairpin motif. The protein assembles a four-OB-fold arrangement by means of symmetric dimerization. In contrast to homotetrameric SSB proteins, asymmetry exists between the two OB folds of D. radiodurans SSB because of sequence differences between the domains. These differences appear to reflect specialized roles that have evolved for each domain. Extensive crystallographic contacts link D. radiodurans SSB dimers in an arrangement that has important implications for higher-order structures of the protein bound to ssDNA. This assembly utilizes the N-terminal OB domain and the ␤-hairpin structure that is unique to Deinococcus and Thermus species SSB proteins. We hypothesize that differences between D. radiodurans SSB and homotetrameric bacterial SSB proteins may confer a selective advantage to D. radiodurans cells that aids viability in environments that challenge genomic stability.I n all organisms, single-stranded DNA (ssDNA)-binding (SSB) proteins sequester and protect ssDNA intermediates that arise during DNA replication, recombination, and repair (1). Their prominent roles in genome maintenance reactions make SSB proteins a requirement for cellular life (2). Although the sequences of SSB family members are highly variable, two common functional themes have emerged that link this class of proteins across evolution. The first is that SSB proteins use a conserved domain called an oligonucleotide͞oligosaccharide-binding (OB) fold to bind ssDNA (3, 4). OB domains bind ssDNA in a cleft formed primarily by ␤-strands, by using aromatic residues that stack against nucleotide bases and positively charged residues that form ionic interactions with the DNA backbone (5-8). The second common feature of cellular SSB proteins is obligate oligomerization that brings together four DNA-binding OB folds. For example, Escherichia coli SSB contains a single OB fold per monomer, but the active form of the protein is a homotetramer with four OB folds (1). This general arrangement appears to define a structural paradigm for bacterial SSB family proteins because all but three of the Ͼ250 currently identifiable bacterial ssb genes encode proteins with a single OB fold.Different...