Structure-functional characterization of Lactococcus AbiA phage defense system.

Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain that is common for many antiviral proteins. AbiA forms a dimer that distinguishes it from AbiK and Abi-P2, which form trimers/hexamers. We show the DNA polymerase activity of AbiA in an in vitro assay and demonstrate that it requires the presence of the HEPN domain which is enzymatically inactive. We validate our biochemical and structural results in vivo through bacteriophage infection assays. Finally, our in vivo results suggest that AbiA-mediated phage defense may not rely on AbiA-mediated cell death.

Zinc Ions Modulate YY1 Activity: Relevance in Carcinogenesis.

YY1 is widely recognized as an intrinsically disordered transcription factor that plays a role in development of many cancers. In most cases, its overexpression is correlated with tumor progression and unfavorable patient outcomes. Our latest research focusing on the role of zinc ions in modulating YY1's interaction with DNA demonstrated that zinc enhances the protein's multimeric state and affinity to its operator. In light of these findings, changes in protein concentration appear to be just one element relevant to modulating YY1-dependent processes. Thus, alterations in zinc ion concentration can directly and specifically impact the regulation of gene expression by YY1, in line with reports indicating a correlation between zinc ion levels and advancement of certain tumors. This review concentrates on other potential consequences of YY1 interaction with zinc ions that may act by altering charge distribution, conformational state distribution, or oligomerization to influence its interactions with molecular partners that can disrupt gene expression patterns.

Mechanism of RecF-RecO-RecR cooperation in bacterial homologous recombination.

In bacteria, one type of homologous-recombination-based DNA-repair pathway involves RecFOR proteins that bind at the junction between single-stranded (ss) and double-stranded (ds) DNA. They facilitate the replacement of SSB protein, which initially covers ssDNA, with RecA, which mediates the search for homologous sequences. However, the molecular mechanism of RecFOR cooperation remains largely unknown. We used Thermus thermophilus proteins to study this system. Here, we present a cryo-electron microscopy structure of the RecF-dsDNA complex, and another reconstruction that shows how RecF interacts with two different regions of the tetrameric RecR ring. Lower-resolution reconstructions of the RecR-RecO subcomplex and the RecFOR-DNA assembly explain how RecO is positioned to interact with ssDNA and SSB, which is proposed to lock the complex on a ssDNA-dsDNA junction. Our results integrate the biochemical data available for the RecFOR system and provide a framework for its complete understanding.

Identification of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein partners using tandem affinity purification and mass spectrometry.

BACKGROUND: According to the World Health Organization Report, depressive disorders affect about 10% of the population. The molecular mechanism of the pathogenesis of depression is still not well understood. The new findings point to phosphatases as potential targets for effective depression therapy. The aim of the present work was the development of a method that would enable the identification of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein partners using a proteomic approach. METHODS: The research was carried out using the PC12 cell line, often used as a model for neurobiological research. The use of the procedure for efficient purification of protein complexes-tandem affinity purification (TAP) will facilitate the identification of proteins interacting with MKP-1, a potential goal of effective antidepressant therapy. RESULTS: Identified proteins belong to various groups: cytoskeletal, ribosomal, nucleic acid binding, chaperones, and enzymes and may potentially be involved in the molecular mechanism of depression. CONCLUSIONS: The presented protocol for the purification of protein complexes is universal and can be successfully used in different mammalian cell lines. Proteins identified in the present work have been reported in the literature concerning studies on depressive disorders, which speaks in favour of their role in depression.

Zinc controls operator affinity of human transcription factor YY1 by mediating dimerization via its N-terminal region.

Human protein Yin Yang 1 (YY1) controls the transcription of hundreds of genes both positively and negatively through interactions with a wide range of partner proteins. Results presented here from proteolytic sensitivity, calorimetry, circular dichroism, fluorescence, NMR, size-exclusion chromatography, SELEX, and EMSA show that purified YY1 forms dimers via its disordered N-terminal region with strong zinc-ion concentration dependence. The YY1 dimer is shown to bind tandem repeats of a canonical recognition DNA sequence with high affinity, and analysis of human YY1 regulatory sites shows that many contain repeats of its recognition elements. YY1 dimerization may compete with partner protein interactions, making control by zinc ion concentration a previously unrecognized factor affecting YY1 gene regulation. Indeed, YY1 is known to be important in many pathogenic processes, including neoplasia, in which zinc ion concentrations are altered. The present results incentivize studies in vivo or in vitro that explore the role of zinc ion concentration in YY1-mediated gene expression.

Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases.

Abortive infection (Abi) is a bacterial antiphage defense strategy involving suicide of the infected cell. Some Abi pathways involve polymerases that are related to reverse transcriptases. They are unique in the way they combine the ability to synthesize DNA in a template-independent manner with protein priming. Here, we report crystal and cryo-electron microscopy structures of two Abi polymerases: AbiK and Abi-P2. Both proteins adopt a bilobal structure with an RT-like domain that comprises palm and fingers subdomains and a unique helical domain. AbiK and Abi-P2 adopt a hexameric and trimeric configuration, respectively, which is unprecedented for reverse transcriptases. Biochemical experiments showed that the formation of these oligomers is required for the DNA polymerization activity. The structure of the AbiK-DNA covalent adduct visualized interactions between the 3' end of DNA and the active site and covalent attachment of the 5' end of DNA to a tyrosine residue used for protein priming. Our data reveal a structural basis of the mechanism of highly unusual template-independent protein-priming polymerases.

The effect of D380Y pathogenic mutation in human Yin Yang 1 on the protein's structure and function.

Yin Yang 1 is a human transcription factor that controls a number of genes and takes part in the regulation of cell cycle, proliferation, differentiation, and neuronal development. Yin Yang 1 is composed of an N-terminal intrinsically disordered fragment and a C-terminal domain responsible for binding to DNA, composed of four zinc fingers. Recently, various alterations in the Yin Yang 1's DNA binding domain were linked with an unexplained intellectual disability named Gabriele-de Vries syndrome. In this paper, a repetitively occurring substitution of aspartate-380 for tyrosine was analyzed to assess its impact on Yin Yang 1's structure and DNA binding. The substitution was found to affect Yin Yang 1's secondary and tertiary structure to a limited extent and to impair the specificity of its interaction with DNA.

RNases H: Structure and mechanism.

RNases H are a family of endonucleases that hydrolyze RNA residues in various nucleic acids. These enzymes are present in all branches of life, and their counterpart domains are also found in reverse transcriptases (RTs) from retroviruses and retroelements. RNases H are divided into two main classes (RNases H1 and H2 or type 1 and type 2 enzymes) with common structural features of the catalytic domain but different range of substrates for enzymatic cleavage. Additionally, a third class is found in some Archaea and bacteria. Besides distinct cellular functions specific for each type of RNases H, this family of proteins is generally involved in the maintenance of genome stability with overlapping and cooperative role in removal of R-loops thus preventing their accumulation. Extensive biochemical and structural studies of RNases H provided not only a comprehensive and complete picture of their mechanism but also revealed key basic principles of nucleic acid recognition and processing. RNase H1 is present in prokaryotes and eukaryotes and cleaves RNA in RNA/DNA hybrids. Its main function is hybrid removal, notably in the context of R-loops. RNase H2, which is also present in all branches of life, can play a similar role but it also has a specialized function in the cleavage of single ribonucleotides embedded in the DNA. RNase H3 is present in Archaea and bacteria and is closely related to RNase H2 in sequence and structure but has RNase H1-like biochemical properties. This review summarizes the mechanisms of substrate recognition and enzymatic cleavage by different classes of RNases H with particular insights into structural features of nucleic acid binding, specificity towards RNA and/or DNA strands and catalysis.

The transcription factor YY2 has less momentous properties of an intrinsically disordered protein than its paralog YY1.

The transcription factor YY2 is a recently discovered paralog of YY1. The two proteins exhibit substantial sequence similarity and partially similar transcriptional activity. They recognize the same DNA sequence in vitro yet bind different promoters in vivo. YY1 comprises two structurally distinct parts: an intrinsically disordered regulatory part and a compact DNA-binding domain. The structure of YY2 is yet unknown. We show that YY2 is structurally similar to YY1, although the conformational state of YY2 is more ordered, as shown by its composition, hydrodynamic properties, spectroscopic signal, and proteolytic susceptibility. As such, YY2's range of molecular partners might be distinct from that of YY1. This could explain different effects of YY1 and YY2 on gene expression patterns and the mechanism of YY proteins in transcriptional regulation.

Improved cytotoxicity of novel TRAIL variants produced as recombinant fusion proteins.

The TNF-Related Apoptosis Inducing Ligand (TRAIL) cytokine triggers apoptosis specifically in cancer cells. Susceptibility of a given cell to TRAIL depends on the activity of regulatory proteins, one of the most important of which is BID. The aim of this study was to increase the cytotoxic potential of TRAIL against cancer cells. TRAIL was fused to the BH3 domain of BID. Hence, TRAIL acted not only as an anticancer agent, but also as a specific carrier for the BID fragment. Two fusion protein variants were obtained by genetic engineering, harboring two different linker sequences. The short linker allowed both parts of the fusion protein to fold into their native structures. The long linker influenced the structure of the fused proteins but nonetheless resulted in their highest cytotoxic activity. Optimal buffer formulation was determined for all the analyzed TRAIL variants. Fusing the BH3 domain of BID to TRAIL improved the cytotoxic potential of TRAIL. Further, these findings may be useful for the optimization of other anticancer drugs based on TRAIL, since the appropriate formulation would secure their native structures during prolonged storage.

Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.

Structural Studies of RNases H2 as an Example of Crystal Structure Determination of Protein-Nucleic Acid Complexes.

RNases H2 are nucleases that cleave nucleic acids that comprise both RNA and DNA. They specifically recognize and cleave junctions between RNA and DNA using an intricate mechanism that involves substrate-assisted catalysis. Archaeal and eukaryotic RNases H2 also cleave the RNA strands of RNA/DNA hybrids. RNases H2 use their activity to maintain the integrity of genetic information. Particularly important is their ability to initiate the removal of single ribonucleotides from genomic DNA. Single ribonucleotides are very common in replicating cells and pose a serious threat to the stability of genomic DNA. The only known pathway for the error-free removal of single ribonucleotides begins with their recognition and cleavage by RNases H2. The importance of these enzymes is further underscored by the fact that mutations in the human enzyme lead to a severe autoimmune disease, Aicardi-Goutières syndrome. This review summarizes methods for the overproduction and purification of bacterial and human RNases H2. We also describe methods for testing the enzymatic activity of these nucleases and their crystallization both in unliganded form and in complex with nucleic acid substrates. We use these studies to describe general principles of the crystallization and structure determination of protein-nucleic acid complexes, particularly for nucleases. As illustrated by the structural studies of RNases H2, such complex structures can reveal intricate and fascinating aspects of the molecular mechanisms of nucleic acid enzymes.

Coordination between the polymerase and RNase H activity of HIV-1 reverse transcriptase.

Replication of human immunodeficiency virus 1 (HIV-1) involves conversion of its single-stranded RNA genome to double-stranded DNA, which is integrated into the genome of the host. This conversion is catalyzed by reverse transcriptase (RT), which possesses DNA polymerase and RNase H domains. The available crystal structures suggest that at any given time the RNA/DNA substrate interacts with only one active site of the two domains of HIV-1 RT. Unknown is whether a simultaneous interaction of the substrate with polymerase and RNase H active sites is possible. Therefore, the mechanism of the coordination of the two activities is not fully understood. We performed molecular dynamics simulations to obtain a conformation of the complex in which the unwound RNA/DNA substrate simultaneously interacts with the polymerase and RNase H active sites. When the RNA/DNA hybrid was immobilized at the polymerase active site, RNase H cleavage occurred, experimentally verifying that the substrate can simultaneously interact with both active sites. These findings demonstrate the existence of a transient conformation of the HIV-1 RT substrate complex, which is important for modulating and coordinating the enzymatic activities of HIV-1 RT.

Physical Interaction of Human Yin Yang 1 Protein with DNA.

Yin Yang 1 (YY1)'s interaction with DNA can result in various, even contradicting, effects on transcription in the form of initiation, activation, or repression. This surprising activity can be explained in the context of the YY1-DNA's complex structure. YY1's DNA-binding domain is formed by four zinc finger motifs. However, the sequence of both the zinc fingers and the linkers is non-canonical, which impairs their docking to the DNA duplex. Short linkers between the zinc fingers impose a concerted binding mechanism. Analysis of the sequences known to be recognized by YY1 suggests different contributions of particular zinc fingers in specific recognition of activated versus repressed promoters. Thermodynamic and kinetic studies show that, although the YY1's N-terminal fragment does not itself bind to DNA, it might regulate the interaction because its presence influences the binding parameters. Meta-analysis of YY1-DNA binding allowed us to observe that YY1's avidity to multiple binding sites is crucial in providing high-affinity specific sequence recognition. Alternatively, other trans-acting factors can modulate the YY1-DNA interaction and influence its outcome. This complex mechanism causes great sensitivity for individual point mutations, an increasing number of which are found in YY1 in cancer tissues.

In vitro fluorescence studies of transcription factor IIB-DNA interaction.

General transcription factor TFIIB is one of the basal constituents of the preinitiation complex of eukaryotic RNA polymerase II, acting as a bridge between the preinitiation complex and the polymerase, and binding promoter DNA in an asymmetric manner, thereby defining the direction of the transcription. Methods of fluorescence spectroscopy together with circular dichroism spectroscopy were used to observe conformational changes in the structure of recombinant human TFIIB after binding to specific DNA sequence. To facilitate the exploration of the structural changes, several site-directed mutations have been introduced altering the fluorescence properties of the protein. Our observations showed that binding of specific DNA sequences changed the protein structure and dynamics, and TFIIB may exist in two conformational states, which can be described by a different microenvironment of W52. Fluorescence studies using both intrinsic and exogenous fluorophores showed that these changes significantly depended on the recognition sequence and concerned various regions of the protein, including those interacting with other transcription factors and RNA polymerase II. DNA binding can cause rearrangements in regions of proteins interacting with the polymerase in a manner dependent on the recognized sequences, and therefore, influence the gene expression.

Intrinsic disorder of human Yin Yang 1 protein.

YY1 (Yin Yang 1) is a zinc finger protein with an essential role in various biological functions via DNA- and protein-protein interactions with numerous partners. YY1 is involved in the regulation of a broad spectrum of cellular processes such as embryogenesis, proliferation, tumorigenesis, and snRNA transcription. The more than 100 reported targets of the YY1 protein suggest that it contains intrinsically disordered regions that are involved in such diverse interactions. Here, we present a study of the structural properties of human YY1 using several biochemical and biophysical techniques (fluorescence, circular dichroism, gel filtration chromatography, proteolytic susceptibility) together with various bioinformatics approaches. To facilitate our exploration of the YY1 structure, the full-length protein as well as an N-terminal fragment (residues 1-295) and the C-terminal DNA binding domain were used. We found the N-terminus to be a non-compact fragment of YY1 with little residual secondary structure and lacking a well-defined tertiary structure. The results of our study indicate that YY1 belongs to the family of intrinsically disordered proteins (IDPs), which exist natively in a partially unfolded conformation.

Crystal structure of RNase H3-substrate complex reveals parallel evolution of RNA/DNA hybrid recognition.

RNases H participate in the replication and maintenance of genomic DNA. RNase H1 cleaves the RNA strand of RNA/DNA hybrids, and RNase H2 in addition hydrolyzes the RNA residue of RNA-DNA junctions. RNase H3 is structurally closely related to RNases H2, but its biochemical properties are similar to type 1 enzymes. Its unique N-terminal substrate-binding domain (N-domain) is related to TATA-binding protein. Here, we report the first crystal structure of RNase H3 in complex with its RNA/DNA substrate. Just like RNases H1, type 3 enzyme recognizes the 2'-OH groups of the RNA strand and detects the DNA strand by binding a phosphate group and inducing B-form conformation. Moreover, the N-domain recognizes RNA and DNA in a manner that is highly similar to the hybrid-binding domain of RNases H1. Our structure demonstrates a remarkable example of parallel evolution of the elements used in the specific recognition of RNA and DNA.

The structural and biochemical characterization of human RNase H2 complex reveals the molecular basis for substrate recognition and Aicardi-Goutières syndrome defects.

RNase H2 cleaves RNA sequences that are part of RNA/DNA hybrids or that are incorporated into DNA, thus, preventing genomic instability and the accumulation of aberrant nucleic acid, which in humans induces Aicardi-Goutières syndrome, a severe autoimmune disorder. The 3.1 Å crystal structure of human RNase H2 presented here allowed us to map the positions of all 29 mutations found in Aicardi-Goutières syndrome patients, several of which were not visible in the previously reported mouse RNase H2. We propose the possible effects of these mutations on the protein stability and function. Bacterial and eukaryotic RNases H2 differ in composition and substrate specificity. Bacterial RNases H2 are monomeric proteins and homologs of the eukaryotic RNases H2 catalytic subunit, which in addition possesses two accessory proteins. The eukaryotic RNase H2 heterotrimeric complex recognizes RNA/DNA hybrids and (5')RNA-DNA(3')/DNA junction hybrids as substrates with similar efficiency, whereas bacterial RNases H2 are highly specialized in the recognition of the (5')RNA-DNA(3') junction and very poorly cleave RNA/DNA hybrids in the presence of Mg(2+) ions. Using the crystal structure of the Thermotoga maritima RNase H2-substrate complex, we modeled the human RNase H2-substrate complex and verified the model by mutational analysis. Our model indicates that the difference in substrate preference stems from the different position of the crucial tyrosine residue involved in substrate binding and recognition.

Contrasting patterns in species boundaries and evolution of anemonefishes (Amphiprioninae, Pomacentridae) in the centre of marine biodiversity.

Many species of coral reef fishes are distinguished by their colour patterns, but genetic studies have shown these are not always good predictors of genetic isolation and species boundaries. The genus Amphiprion comprises several species that have very similar colouration. Additionally, morphological characters are so variable, that sibling species can show a considerable overlap, making it difficult to differentiate them. In this study, we investigated the species boundaries between the sibling species pair A. ocellaris and A. percula (Subgenus Actinicola) and three closely related species of the subgenus Phalerebus (A. akallopisos, A. perideraion, A. sandaracinos) by phylogenetic analysis of mitochondrial cytochrome b and control region sequences. These two subgenera show strong differences in their patterns of species boundaries. Within the A. ocellaris/A. percula complex, five clades were found representing different geographic regions. Two major divergences both with genetic distances of 4-7% in cty b and 17-19% in the d-loop region indicate the presence of three instead of two deep evolutionary lineages. The species of the subgenus Phalerebus show three monophyletic clades, independent of the geographical location of origin, but concordant to the morphological species classification. The genetic distances between the Phalerebus species were 2-5% in cty b and 10-12% in the control region.