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RECQ1 is the shortest among the five human RecQ helicases comprising of two RecA like domains, a zinc-binding domain and a RecQ C-terminal domain containing the winged-helix (WH). Mutations or deletions on the tip of a β-hairpin located in the WH domain are known to abolish the unwinding activity. Interestingly, the same mutations on the β-hairpin of annealing incompetent RECQ1 mutant (RECQ1T1) have been reported to restore its annealing activity. In an attempt to unravel the strand annealing mechanism, we have crystallized a fragment of RECQ1 encompassing D2–Zn–WH domains harbouring mutations on the β-hairpin. From our crystal structure data and interface analysis, we have demonstrated that an α-helix located in zinc-binding domain potentially interacts with residues of WH domain, which plays a significant role in strand annealing activity. We have shown that deletion of the α-helix or mutation of specific residues on it restores strand annealing activity of annealing deficient constructs of RECQ1. Our results also demonstrate that mutations on the α-helix induce conformational changes and affects DNA stimulated ATP hydrolysis and unwinding activity of RECQ1. Our study, for the first time, provides insight into the conformational requirements of the WH domain for efficient strand annealing by human RECQ1.

Intrinsically disordered protein (IDP) sequences often contain a high proportion of charged residues in conjunction with their high degree of hydrophilicity and solvation. For high net charge IDPs, long-range electrostatic interactions are thought to play a role in modulating the strength or kinetics of protein-protein interactions. In this work, we examined intramolecular interactions mediated by charged regions of a model IDP, the C-terminal tail of the phosphatase Fcp1. Specifically, this work focuses on intermolecular interactions between acidic and basic patches in the primary structure of Fcp1 and their contributions to binding its predominantly basic partner, the winged helix domain of Rap74. We observe both intramolecular and intermolecular interactions through paramagnetic relaxation enhancement (PRE) consistent with oppositely charged regions associating with one another, both in unbound Fcp1 and in the Fcp1-Rap74 complex. Formation of this complex is strongly driven by hydrophobic interactions in the minimal binding motif. Here, we test the hypothesis that charged residues in Fcp1 that flank the binding helix also contribute to the strength of binding. Charge inversion mutations in Fcp1 generally support this hypothesis, while PRE data suggest substitution of observed transient interactions in the unbound ensemble for similarly transient interactions with Rap74 in the complex.

RNA polymerase III (Pol III) transcribes essential structured small RNAs, such as tRNAs, 5S rRNA and U6 snRNA. The transcriptional activity of Pol III is tightly controlled and its dysregulation is associated with human diseases, such as cancer. Human Pol III has two isoforms with difference only in one of its subunits RPC7 (α and β). Despite structural studies of yeast Pol III, structure of human Pol III remains unsolved. Here, we determined the structures of 17-subunit human Pol IIIα complex in the backtracked and post-translocation states, respectively. Human Pol III contains a generally conserved catalytic core, similar to that of yeast counterpart, and structurally unique RPC3-RPC6-RPC7 heterotrimer and RPC10. The N-ribbon of TFIIS-like RPC10 docks on the RPC4-RPC5 heterodimer and the C-ribbon inserts into the funnel of Pol III in the backtracked state but is more flexible in the post-translocation state. RPC7 threads through the heterotrimer and bridges the stalk and Pol III core module. The winged helix 1 domain of RPC6 and the N-terminal region of RPC7α stabilize each other and may prevent Maf1-mediated repression of Pol III activity. The C-terminal FeS cluster of RPC6 coordinates a network of interactions that mediate core-heterotrimer contacts and stabilize Pol III. Our structural analysis sheds new light on the molecular mechanism of human Pol IIIα-specific transcriptional regulation and provides explanations for upregulated Pol III activity in RPC7α-dominant cancer cells.

Origin licensing ensures precise once per cell cycle replication in eukaryotic cells. The Origin Recognition Complex, Cdc6 and Cdt1 load Mcm2-7 helicase (MCM) into a double hexamer, bound around duplex DNA. The complex formed by ORC-Cdc6 bound to duplex DNA (OC) recruits the MCM-Cdt1 complex into the replication origins. Through the stacking of both complexes, the duplex DNA is inserted inside the helicase by an unknown mechanism. In this paper we show that the DNA insertion comes with a topological problem in the stacking of OC with MCM-Cdt1. Unless an essential, conserved C terminal winged helix domain (C-WHD) of Cdt1 is present, the MCM splits into two halves. The binding of this domain with the essential C-WHD of Mcm6, allows the latching between the MCM-Cdt1 and OC, through a conserved Orc5 AAA-lid interaction. Our work provides new insights into how DNA is inserted into the eukaryotic replicative helicase, through a series of synchronized events., During pre-Replication Complex, eukaryotic cells load two MCMs into a head-to-head Double Hexamer around duplex DNA (DH). Here the authors preRC assembly assay with purified proteins to reveal insights into S. cerevisiae’s first steps that lead to the DH formation.

Maf1 is a conserved inhibitor of RNA polymerase III (Pol III) that influences phenotypes ranging from metabolic efficiency to lifespan. Here, we present a 3.3-A-resolution cryo-EM structure of yeast Maf1 bound to Pol III, establishing that Maf1 sequesters Pol III elements involved in transcription initiation and binds the mobile C34 winged helix 2 domain, sealing off the active site. The Maf1 binding site overlaps with that of TFIIIB in the preinitiation complex. A 3.3-A-resolution cryo-EM structure of yeast Maf1 bound to RNA polymerase III (Pol III) explains the molecular mechanism for Pol III inhibition.

Domains are the structural, functional and evolutionary units of proteins. They combine to form multidomain proteins. The evolutionary history of this molecular combinatorics has been studied with phylogenomic methods. Here, we construct networks of domain organization and explore their evolution. A time series of networks revealed two ancient waves of structural novelty arising from ancient ‘p-loop’ and ‘winged helix’ domains and a massive ‘big bang’ of domain organization. The evolutionary recruitment of domains was highly modular, hierarchical and ongoing. Domain rearrangements elicited non-random and scale-free network structure. Comparative analyses of preferential attachment, randomness and modularity showed yin-and-yang complementary transition and biphasic patterns along the structural chronology. Remarkably, the evolving networks highlighted a central evolutionary role of cofactor-supporting structures of non-ribosomal peptide synthesis pathways, likely crucial to the early development of the genetic code. Some highly modular domains featured dual response regulation in two-component signal transduction systems with DNA-binding activity linked to transcriptional regulation of responses to environmental change. Interestingly, hub domains across the evolving networks shared the historical role of DNA binding and editing, an ancient protein function in molecular evolution. Our investigation unfolds historical source-sink patterns of evolutionary recruitment that further our understanding of protein architectures and functions.

Funding: Open Access publication fees were provided by the University of St Andrews. The eukaryotic single-stranded DNA binding factor replication protein A (RPA) is essential for DNA replication, repair and recombination. RPA is a heterotrimer containing six related OB folds and a winged helix-turn-helix (wH) domain. The OB folds are designated DBD-A through DBD-F, with DBD-A through DBD-D being directly involved in ssDNA binding. DBD-C is located at the C-terminus of the RPA1 protein and has a distinctive structure that includes an integral C4 zinc finger, while the wH domain is found at the C-terminus of the RPA2 protein. Previously characterised archaeal RPA proteins fall into a number of classes with varying numbers of OB folds, but one widespread class includes proteins that contain a C4 or C3H zinc finger followed by a 100-120 amino acid C-terminal region reported to lack detectable sequence or structural similarity. Here, the sequences spanning this zinc finger and including the C-terminal region are shown to comprise a previously unrecognised DBD-C-like OB fold, confirming the evolutionary relatedness of this group of archaeal RPA proteins to eukaryotic RPA1. The evolutionary relationship between eukaryotic and archaeal RPA is further underscored by the presence of RPA2-like proteins comprising an OB fold and C-terminal winged helix (wH) domain in multiple species and crucially, suggests that several biochemically characterised archaeal RPA proteins previously thought to exist as monomers are likely to be RPA1-RPA2 heterodimers. Publisher PDF

Cockayne syndrome group B protein (CSB), a member of the SWI/SNF superfamily, resides in an elongating RNA polymerase II (RNAPII) complex and regulates transcription elongation. CSB contains a C-terminal winged helix domain (WHD) that binds to ubiquitin and plays an important role in DNA repair. However, little is known about the role of the CSB-WHD in transcription regulation. Here, we report that CSB is dependent upon its WHD to regulate RNAPII abundance at promoter proximal pause (PPP) sites of several actively transcribed genes, a key step in the regulation of transcription elongation. We show that two ubiquitin binding-defective mutations in the CSB-WHD, which impair CSB’s ability to promote cell survival in response to treatment with cisplatin, have little impact on its ability to stimulate RNAPII occupancy at PPP sites. In addition, we demonstrate that two cancer-associated CSB mutations, which are located on the opposite side of the CSB-WHD away from its ubiquitin-binding pocket, impair CSB’s ability to promote RNAPII occupancy at PPP sites. Taken together, these results suggest that CSB promotes RNAPII association with PPP sites in a manner requiring the CSB-WHD but independent of its ubiquitin-binding activity. These results further imply that CSB-mediated RNAPII occupancy at PPP sites is mechanistically separable from CSB-mediated repair of cisplatin-induced DNA damage.

15 p.-6 fig., An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA-protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin., National Science Centre [2012/04/A/NZ1/00048 to I.K.;2017/26/D/NZ1/00239 to K.W.]; Foundation for Polish Science [TEAM, POIR.04.04.00-00-5C75/17-00 to I.K.]; International Institute of Molecular and Cell Biology in Warsaw (to J.M.B.); Ministerio de Economía,Industria y Competitividad (MINECO/AEI) [BIO2012-30852, RTI2018-094549-B-I00 to R.G.]. Funding for open access charge: Foundation for Polish Science [TEAM,POIR.04.04.00-00-5C75/17-00].

Background Foxc2 is a member of the winged helix/forkhead (Fox) box family of transcription factors. Loss of function of Foxc2 causes craniofacial abnormalities such as cleft palate and deformed cranial base, but its role during craniofacial development remains to be elucidated RESULTS: The contributions of Foxc2-positive and its descendant cells to the craniofacial structure at E18.5 were examined using a tamoxifen-inducible Cre driver mouse (Foxc2-CreERT2) crossed with the R26R-LacZ reporter mouse. Foxc2 expression at E8.5 is restricted to the cranial mesenchyme, contributing to specific components including the cranial base, sensory capsule, tongue, upper incisor, and middle ear. Expression at E10.5 was still positively regulated in most of those regions. In situ hybridization analysis of Foxc2 and its closely related gene, Foxc1, revealed that expression domains of these genes largely overlap in the cephalic mesenchyme. Meanwhile, the tongue expressed Foxc2 but not Foxc1, and its development was affected by the neural crest-specific deletion of Foxc2 in mice (Wnt1-Cre; Foxc2fl/fl ). Conclusions Foxc2 is expressed in cranial mesenchyme that contributes to specific craniofacial tissue components from an early stage, and it seems to be involved in their development in cooperation with Foxc1. Foxc2 also has its own role in tongue development. This article is protected by copyright. All rights reserved.

Forkhead box G1 (FOXG1) is a transcription factor mainly expressed in the brain that plays a critical role in the development and regionalization of the forebrain. Aberrant expression of FOXG1 has implications in FOXG1 syndrome, a serious neurodevelopmental disorder. Here, we report the crystal structure of the FOXG1 DNA-binding domain (DBD) in complex with the forkhead consensus DNA site DBE2 at the resolution of 1.6 A. FOXG1-DBD adopts a typical winged helix fold. Compared to those of other FOX-DBD/DBE2 structures, the N terminus, H3 helix and wing2 region of FOXG1-DBD exhibit differences in DNA recognition. The FOXG1-DBD wing2 region adopts a unique architecture composed of two β-strands that differs from all other known FOX-DBD wing2 folds. Mutation assays revealed that the disease-causing mutations within the FOXG1-DBD affect DNA binding, protein thermal stability, or both. Our report provides initial insight into how FOXG1 binds DNA and sheds light on how disease-causing mutations in FOXG1-DBD affect its DNA-binding ability.

Minichromosome maintenance 8 (MCM8) is a recently identified member of the minichromosome maintenance family, which possesses helicase and ATPase activity. It interacts with MCM9 and participates in homologous recombination repair. The structure of MCM8 is unclear now. Here, we report the crystal structure of the winged-helix domain of human MCM8 (MCM8-WHD) at 1.21 A resolution. MCM8-WHD adopts a conserved winged-helix architecture. Structure analysis and biochemical study results showed the DNA binding ability and crucial residues of MCM8-WHD. Our results are helpful to understand the function of MCM8.

Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation. Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provide roles for previously identified UAF domains that include the Rrn5 SANT and histone fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal winged helix domain and a disordered tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual-function UAF complex.

Bacterial xenogeneic silencers play important roles in bacterial evolution by recognizing and inhibiting expression from foreign genes acquired through horizontal gene transfer, thereby buffering against potential fitness consequences of their misregulated expression. Here, the detailed DNA binding properties of Rok, a xenogeneic silencer in Bacillus subtilis, was studied using protein binding microarray, and the solution structure of its C-terminal DNA binding domain was determined in complex with DNA. The C-terminal domain of Rok adopts a typical winged helix fold, with a novel DNA recognition mechanism different from other winged helix proteins or xenogeneic silencers. Rok binds the DNA minor groove by forming hydrogen bonds to bases through N154, T156 at the N-terminal of α3 helix and R174 of wing W1, assisted by four lysine residues interacting electrostatically with DNA backbone phosphate groups. These structural features endow Rok with preference towards DNA sequences harboring AACTA, TACTA, and flexible multiple TpA steps, while rigid A-tracts are disfavored. Correspondingly, the Bacillus genomes containing Rok are rich in A-tracts and show a dramatic underrepresentation of AACTA and TACTA, which are significantly enriched in Rok binding regions. These observations suggest that the xenogeneic silencing protein and its resident genome may have evolved cooperatively.

The ATP-dependent chromatin remodeler CSB is implicated in a variety of different DNA repair mechanisms, including transcription-coupled nucleotide excision repair (TC-NER), base excision repair and DNA double strand break (DSB) repair. However, how CSB is regulated in these various repair processes is not well understood. Here we report that the first 30 amino acids of CSB along with two phosphorylation events on S10 and S158, previously reported to be required for CSB function in homologous recombination (HR)-mediated repair, are dispensable for repairing UV-induced DNA damage, suggesting that the regulation of CSB in these two types of repair are carried out by distinct mechanisms. In addition, we show that although the central ATPase domain of CSB is engaged in interactions with both the N- and C-terminal regions, these interactions are disrupted following UV-induced DNA damage. The UV-induced disengagement of the C-terminal region of CSB from the ATPase domain requires two conserved amino acids W1486 and L1488, which are thought to contribute to the hydrophobic core formation of the winged helix domain (WHD) at its C-terminus. Failure to undergo UV-induced dissociation of the C-terminal region of CSB from the ATPase domain is associated with impairment in its UV-induced chromatin association, its UV-induced post-translational modification as well as cell survival. Collectively, these findings suggest that UV-induced dissociation of CSB domain interactions is a necessary step in repairing UV-induced DNA damage and that the WHD of CSB plays a key role in this dissociation.

Transcription factors (TFs) participate in a wide range of cellular processes due to their inherent function as essential regulatory proteins. Their dysfunction has been linked to numerous human diseases. The forkhead box (FOX) family of TFs belongs to the “winged helix” superfamily, consisting of proteins sharing a related winged helix-turn-helix DNA-binding motif. FOX genes have been extensively present during vertebrates and invertebrates’ evolution, participating in numerous molecular cascades and biological functions, such as embryonic development and organogenesis, cell cycle regulation, metabolism control, stem cell niche maintenance, signal transduction, and many others. FOXD1, a forkhead TF, has been related to different key biological processes such as kidney and retina development and embryo implantation. FOXD1 dysfunction has been linked to different pathologies, thereby constituting a diagnostic biomarker and a promising target for future therapies. This paper aims to present, for the first time, a comprehensive review of FOXD1’s role in mouse development and human disease. Molecular, structural, and functional aspects of FOXD1 are presented in light of physiological and pathogenic conditions, including its role in human disease aetiology, such as cancer and recurrent pregnancy loss. Taken together, the information given here should enable a better understanding of FOXD1 function for basic science researchers and clinicians. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature.

Mycobacterial Lhr is a DNA damage-inducible superfamily 2 helicase that uses adenosine triphosphate (ATP) hydrolysis to drive unidirectional 3′-to-5′ translocation along single-stranded DNA (ssDNA) and to unwind RNA:DNA duplexes en route. ATPase, translocase and helicase activities are encompassed within the N-terminal 856-amino acid segment. The crystal structure of Lhr-(1–856) in complex with AMPPNP•Mg2+ and ssDNA defines a new helicase family. The enzyme comprises two N-terminal RecA-like modules, a winged helix (WH) domain and a unique C-terminal domain. The 3′ ssDNA end binds in a crescent-shaped groove at the interface between the first RecA domain and the WH domain and tracks 5′ into a groove between the second RecA and C domains. A kissing interaction between the second RecA and C domains forms an aperture that demarcates a putative junction between the loading strand tail and the duplex, with the first duplex nucleoside bookended by stacking on Trp597. Intercalation of Ile528 between nucleosides of the loading strand creates another bookend. Coupling of ATP hydrolysis to RNA:DNA unwinding is dependent on Trp597 and Ile528, and on Thr145 and Arg279 that contact phosphates of the loading strand. The structural and functional data suggest a ratchet mechanism of translocation and unwinding coupled to ATP-driven domain movements.

RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency, and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a RecQ C-terminal region (RQC) found in the other RecQ paralogues; such a region consists of a zinc domain and a winged helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. Inductively coupled plasma-atomic emission spectrometry detected the unusual presence of two zinc clusters within the zinc domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative zinc ligands and the aromatic residue predicted to be at the tip of the winged helix β-hairpin), showed a decrease in DNA binding, unwinding, and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.

Maf1 is a highly conserved central regulator of transcription by RNA polymerase III (Pol III), and Maf1 activity influences a wide range of phenotypes from metabolic efficiency to lifespan. Here, we present a 3.3 Å cryo-EM structure of yeast Maf1 bound to Pol III, which establishes how Maf1 achieves transcription repression. In the Maf1-bound state, Pol III elements that are involved in transcription initiation are sequestered, and the active site is sealed off due to ordering of the mobile C34 winged helix 2 domain. Specifically, the Maf1 binding site overlaps with the binding site of the Pol III transcription factor TFIIIB and DNA in the pre-initiation complex, rationalizing that binding of Maf1 and TFIIIB to Pol III are mutually exclusive. We validate our structure using variants of Maf1 with impaired transcription-inhibition activity. These results reveal the exact mechanism of Pol III inhibition by Maf1, and rationalize previous biochemical data.

Made available in DSpace on 2020-12-10T19:36:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-10-01 Fundacao Araucaria Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The DNA/RNA-binding KIN protein was discovered in 1989, and since then, it has been found to participate in several processes, e.g., as a transcription factor in bacteria, yeasts, and plants, in immunoglobulin isotype switching, and in the repair and resolution of double-strand breaks caused by ionizing radiation. However, the complete three-dimensional structure and biophysical properties of KIN remain important information for clarifying its function and to help elucidate mechanisms associated with it not yet completely understood. The present study provides data on phylogenetic analyses of the different domains, as well as a biophysical characterization of the human KIN protein (HSAKIN) using bioinformatics techniques, circular dichroism spectroscopy, and differential scanning calorimetry to estimate the composition of secondary structure elements; further studies were performed to determine the biophysical parameters Delta H-m and T-m. The phylogenetic analysis indicated that the zinc-finger and winged helix domains are highly conserved in KIN, with mean identity of 90.37% and 65.36%, respectively. The KOW motif was conserved only among the higher eukaryotes, indicating that this motif emerged later on the evolutionary timescale. HSAKIN has more than 50% of its secondary structure composed by random coil and beta-turns. The highest values of Delta H-m and T-m were found at pH 7.4 suggesting a stable structure at physiological conditions. The characteristics found for HSAKIN are primarily due to its relatively low composition of alpha-helices and beta-strands, making up less than half of the protein structure. Univ Estadual Maringa, Dept Technol, Av Angelo Moreira da Fonseca 1800, BR-87506370 Umuarama, PR, Brazil Univ Estadual Paulista, Inst Biociencias Letras & Ciencias Exatas, Dept Phys, Sao Jose Do Rio Preto, SP, Brazil Univ Estadual Maringa, Dept Biotechnol Genet & Cell Biol, Maringa, Parana, Brazil Univ Estadual Maringa, Ctr Mol Struct & Funct Biol, CBM Res Support Ctr Complex, Maringa, Parana, Brazil Univ Fed Parana, Dept Biochem, Curitiba, Parana, Brazil Univ Estadual Paulista, Inst Biociencias Letras & Ciencias Exatas, Dept Phys, Sao Jose Do Rio Preto, SP, Brazil Fundacao Araucaria: 147/14 Fundacao Araucaria: 40/16 CAPES: 001 CNPq: 305960/2015-6

Cockayne syndrome group B (CSB, also known as ERCC6) protein is involved in many DNA repair processes and essential for transcription-coupled repair (TCR). The central region of CSB has the helicase motif, whereas the C-terminal region contains important regulatory elements for repair of UV- and oxidative stress-induced damages and double-strand breaks (DSBs). A previous study suggested that a small part (∼30 residues) within this region was responsible for binding to ubiquitin (Ub). Here, we show that the Ub-binding of CSB requires a larger part of CSB, which was previously identified as a winged-helix domain (WHD) and is involved in the recruitment of CSB to DSBs. We also present the crystal structure of CSB WHD in complex with Ub. CSB WHD folds as a single globular domain, defining a class of Ub-binding domains (UBDs) different from 23 UBD classes identified so far. The second α-helix and C-terminal extremity of CSB WHD interact with Ub. Together with structure-guided mutational analysis, we identified the residues critical for the binding to Ub. CSB mutants defective in the Ub binding reduced repair of UV-induced damage. This study supports the notion that DSB repair and TCR may be associated with the Ub-binding of CSB.

International audience; DprA, a ubiquitous multidomain DNA-binding protein, plays a crucial role during natural transformation in bacteria. Here, we carried out the structural analysis of DprA from the human pathogen Helicobacter pylori by combining data issued from the 1.8 Å resolution X-ray structure of the Pfam02481 domain dimer (RF), the NMR structure of the carboxy-terminal domain (CTD) and the low-resolution structure of the full-length DprA dimer obtained in solution by SAXS. In particular, we sought a molecular function for the CTD, a domain that we show here is essential for transformation in H. pylori. Albeit its structural homology to winged helix DNA binding motifs, we confirmed that the isolated CTD does not interact with ssDNA nor with dsDNA. The key R52 and K137 residues of RF are crucial for these two interactions. Search for sequences harboring homology to either HpDprA or Rhodopseudomonas palustris DprA CTDs led to the identification of conserved patches in the two CTD. Our structural study revealed the similarity of the structures adopted by these residues in RpDprA CTD and HpDprA CTD. This argues for a conserved, but yet to be defined, CTD function, distinct from DNA binding. This article is protected by copyright. All rights reserved.