Cdt1 Self-associates via the Winged-Helix Domain of the Central Region during the Licensing Reaction, Which Is Inhibited by Geminin.

The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein-protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein-protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two ß-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.

Dissection of Functional Domains of Orc1-2, the Archaeal Global DNA Damage-Responsive Regulator.

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

The Bacillus subtilis PriA Winged Helix Domain Is Critical for Surviving DNA Damage.

DNA replication forks regularly encounter lesions or other impediments that result in a blockage to fork progression. PriA is one of the key proteins used by virtually all eubacteria to survive conditions that result in a blockage to replication fork movement. PriA directly binds stalled replication forks and initiates fork restart allowing for chromosomes to be fully duplicated under stressful conditions. We used a CRISPR-Cas gene editing approach to map PriA residues critical for surviving DNA damage induced by several antibiotics in B. subtilis. We find that the winged helix (WH) domain in B. subtilis PriA is critical for surviving DNA damage and participates in DNA binding. The important in vivo function of the WH domain mapped to distinct surfaces that were also conserved among several Gram-positive human pathogens. In addition, we identified an amino acid linker neighboring the WH domain that is greatly extended in B. subtilis due to an insertion. Shortening this linker induced a hypersensitive phenotype to DNA damage, suggesting that its extended length is critical for efficient replication fork restart in vivo. Because the WH domain is dispensable in E. coli PriA, our findings demonstrate an important difference in the contribution of the WH domain during fork restart in B. subtilis. Furthermore, with our results we suggest that this highly variable region in PriA could provide different functions across diverse bacterial organisms. IMPORTANCE PriA is an important protein found in virtually all bacteria that recognizes stalled replication forks orchestrating fork restart. PriA homologs contain a winged helix (WH) domain. The E. coli PriA WH domain is dispensable and functions in a fork restart pathway that is not conserved outside of E. coli and closely related proteobacteria. We analyzed the importance of the WH domain and an associated linker in B. subtilis and found that both are critical for surviving DNA damage. This function mapped to a small motif at the C-terminal end of the WH domain, which is also conserved in pathogenic bacteria. The motif was not required for DNA binding and therefore may perform a novel function in the replication fork restart pathway.

Remote Homology Detection Identifies a Eukaryotic RPA DBD-C-like DNA Binding Domain as a Conserved Feature of Archaeal Rpa1-Like Proteins.

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.

The Winged Helix Domain of CSB Regulates RNAPII Occupancy at Promoter Proximal Pause Sites.

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.

Crystal structure of the winged-helix domain of MCM8.

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 Å 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.

Structures of the Mitochondrial CDP-DAG Synthase Tam41 Suggest a Potential Lipid Substrate Pathway from Membrane to the Active Site.

In mitochondria, CDP-diacylglycerol (CDP-DAG) is a crucial precursor for cardiolipin biosynthesis. Mitochondrial CDP-DAG is synthesized by the translocator assembly and maintenance protein 41 (Tam41) through an elusive process. Here we show that Tam41 adopts sequential catalytic mechanism, and report crystal structures of the bulk N-terminal region of Tam41 from Schizosaccharomyces pombe in the apo and CTP-bound state. The structure reveals that Tam41 contains a nucleotidyltransferase (NTase) domain and a winged helix domain. CTP binds to an "L"-shaped pocket sandwiched between the two domains. Rearrangement of a loop region near the active site is essential for opening the CTP-binding pocket. Docking of phosphatidic acid/CDP-DAG in the structure suggests a lipid entry/exit pathway connected to the "L"-shaped pocket. The C-terminal region of SpTam41 contains a positively charged amphipathic helix crucial for membrane association and participates in binding phospholipids. These results provide detailed insights into the mechanism of CDP-DAG biosynthesis in mitochondria.

Efficient UV repair requires disengagement of the CSB winged helix domain from the CSB ATPase domain.

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.

Functions of the TFIIE-Related Tandem Winged-Helix Domain of Rpc34 in RNA Polymerase III Initiation and Elongation.

Rpc34 is a subunit of the Rpc82/34/31 subcomplex residing on the DNA-binding cleft of RNA polymerase (Pol) III. Rpc34 contains a structurally flexible N-terminal tandem winged-helix (tWH) domain related to the TFIIE transcription factor. While the second WH (WH2) fold of the tWH domain is known to function in DNA melting activity during transcription initiation, the functional role of the WH1 fold is unknown. In this study, we generated a series of new Rpc34 tWH mutants conferring a cold-sensitive growth phenotype. We found that the tWH mutations severely compromised in vitro transcription activity due to destabilization of the preinitiation complex (PIC). Site-specific protein photo-cross-linking analysis indicated that the tWH domain persistently interacts with protein subunits of the Pol III cleft in the PIC and the ternary elongation complex (TEC). Furthermore, purified Pol III proteins with tWH mutations also showed reduced efficiency in RNA elongation. Our study results suggest that the tWH domain is an important protein module above the Pol III cleft that integrates protein and nucleic acid interactions for initiation and elongation.

HSPC280, a winged helix protein expressed in the subventricular zone of the developing ganglionic eminences, inhibits neuronal differentiation.

Winged helix proteins have critical roles in a variety of developmental processes. During a screening for genes expressed in the developing forebrain, we identified HSPC280, a non-typical winged helix protein, which shares similarity with a protein-protein interaction domain found in the proteins of the actin-binding Rho-activating protein family. In this work, we analyzed HSPC280 expression during mouse development as well as during neuronal differentiation of mouse Neuro2a cells. HSPC280 expression is tightly regulated; during mouse development, it was detected predominantly in the ganglionic eminences of the ventral telencephalon, from their appearance at E11.5 to P0, with the highest levels between E13.5 and E15.5, a period that correlates with the peak of neurogenesis in these structures. Comparative expression analysis of HSPC280 with Dlx2, cyclinD2 and Lhx6 revealed that, within the ganglionic eminences, HSPC280 was restricted in the proliferating cell population of the subventricular zone, in a pattern similar to that of cyclinD2. Finally, we showed that HSPC280 is a nuclear protein which, when overexpressed in Neuro2a cells, it inhibited neuronal differentiation in vitro, suggesting its involvement in the mechanisms controlling neural progenitor cells proliferation.

Distinct structural features of Rex-family repressors to sense redox levels in anaerobes and aerobes.

The Rex-family repressors sense redox levels by alternative binding to NADH or NAD(+). Unlike other Rex proteins that regulate aerobic respiration, RSP controls ethanol fermentation in the obligate anaerobe Thermoanaerobacter ethanolicus JW200(T). It is also found in other anaerobic microorganisms. Here we present the crystal structures of apo-RSP, RSP/NADH and RSP/NAD(+)/DNA, which are the first structures of Rex-family members from an obligate anaerobe. RSP functions as a homodimer. It assumes an open conformation when bound to the operator DNA and a closed conformation when not DNA-bound. The DNA binds to the N-terminal winged-helix domain and the dinucleotide, either reduced or oxidized, binds to the C-terminal Rossmann-fold domain. The two distinct orientations of nicotinamide ring, anti in NADH and syn in NAD(+), give rise to two sets of protein-ligand interactions. Consequently, NADH binding makes RSP into a closed conformation, which does not bind to DNA. Both the conserved residues and the DNA specificity of RSP show a number of variations from those of the aerobic Rex, reflecting different structural bases for redox-sensing by the anaerobic and aerobic Rex-family members.

Sequential protein NMR assignments in the liquid state via sequential data acquisition.

Two different NMR pulse schemes involving sequential (1)H data acquisition are presented for achieving protein backbone sequential resonance assignments: (i) acquisition of 3D {HCCNH and HNCACONH} and (ii) collection of 3D {HNCOCANH and HNCACONH} chemical shift correlation spectra using uniformly (13)C,(15)N labelled proteins. The sequential acquisition of these spectra reduces the overall experimental time by a factor of ≈2 as compared to individual acquisitions. The suitability of this approach is experimentally demonstrated for the C-terminal winged helix (WH) domain of the minichromosome maintenance (MCM) complex of Sulfolobus solfataricus.

Mapping the spectrum of conformational states of the DNA- and C-gates in Bacillus subtilis gyrase.

Type II DNA topoisomerases alter the supercoiling state of DNA in an ATP-dependent fashion that requires large conformational changes. The directionality of DNA strand transfer is controlled by three transient protein interfaces, termed the N-gate, DNA-gate, and C-gate. Bacterial gyrase is a type II DNA topoisomerase of A2B2 composition. The N-gate is formed by the two GyrB subunits and the GyrA subunits form the DNA- and C-gates. In structures of type II topoisomerase fragments, the DNA- and C-gates delimit a cavity for DNA and can be open or closed. However, the conformational space accessible has not yet been mapped. Here, we describe the crystal structure of the Bacillus subtilis DNA gyrase A subunit lacking the C-terminal DNA-wrapping domains. Five dimeric states of the GyrA N-terminal domain are observed, with their DNA- and C-gates either closed, or open to different extents. All of these conformations can in principle accommodate double-stranded DNA in the central cavity but only one conformation has its DNA-gate open wide enough for DNA to enter. The structure thus reflects the lower limit of DNA-gate opening that must occur during gyrase catalysis. The DNA-gate is formed by two flat surfaces, with few interactions. In contrast, the C-gate exhibits a highly undulated surface and forms a large number of interactions. None of the dimers in the crystal structures display an open C-gate that would allow DNA passage, in agreement with a transient opening of this gate during the catalytic cycle of DNA supercoiling.

Structure of a virulence regulatory factor CvfB reveals a novel winged helix RNA binding module.

CvfB is a conserved regulatory protein important for the virulence of Staphylococcus aureus. We show here that CvfB binds RNA. The crystal structure of the CvfB ortholog from Streptococcus pneumoniae at 1.4 A resolution reveals a unique RNA binding protein that is formed from a concatenation of well-known structural modules that bind nucleic acids: three consecutive S1 RNA binding domains and a winged helix (WH) domain. The third S1 and the WH domains are required for cooperative RNA binding and form a continuous surface that likely contributes to the RNA interaction. The WH domain is critical to CvfB function and contains a unique sequence motif. Thus CvfB represents a novel assembly of modules for binding RNA.