Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein.     

A Replication Origin of Bacillus thuringiensis

A replication origin of Bacillus thuringiensis (Bt) was found in a Bacillus thuringiensis–Escherichia coli shuttle vector of pHT3101. Deletion analysis showed that the replication origin was segregationally stable at suitable temperature for Bt growth. The fragment containing the replication origin was cloned in pUC18 and sequenced. It was 261 base pairs in length, located in the open reading frame 2 (ORF2) of BTSPB sequence. The 261-bp fragment was cloned in pBR322, creating an improved Bt-E. coli shuttle vector pBR261, which contained two resistance genes responsible for ampicillin and tetracycline. Our study showed that the replication origin structure could be recognized by replication protein of host cells. Received: 26 July 1999 / Accepted: 30 August 1999     

Relation of the Segregative Origin of Chromosome Replication to the Origin of Replication After Amino Acid Starvation

Cultures of Escherichia coli 15T(-) and K-12 were labeled with (3)H-thymine before, during, and after amino acid starvation. The number of labeled segregating units was measured by autoradiography of microcolonies derived from the labeled cells. In both strains, labels inserted before starvation and during starvation appeared to segregate as if incorporated into the same polynucleotide strands. However, labels inserted during and after starvation segregated as if incorporated into different polynucleotide strands. In view of previous data, it was concluded that replication after amino acid starvation originates from the region of the chromosome which serves as the origin for replication during normal growth and division.     

Mutational Robustness Accelerates the Origin of Novel RNA Phenotypes through Phenotypic Plasticity

Novel phenotypes can originate either through mutations in existing genotypes or through phenotypic plasticity, the ability of one genotype to form multiple phenotypes. From molecules to organisms, plasticity is a ubiquitous feature of life, and a potential source of exaptations, adaptive traits that originated for nonadaptive reasons. Another ubiquitous feature is robustness to mutations, although it is unknown whether such robustness helps or hinders the origin of new phenotypes through plasticity. RNA is ideal to address this question, because it shows extensive plasticity in its secondary structure phenotypes, a consequence of their continual folding and unfolding, and these phenotypes have important biological functions. Moreover, RNA is to some extent robust to mutations. This robustness structures RNA genotype space into myriad connected networks of genotypes with the same phenotype, and it influences the dynamics of evolving populations on a genotype network. In this study I show that both effects help accelerate the exploration of novel phenotypes through plasticity. My observations are based on many RNA molecules sampled at random from RNA sequence space, and on 30 biological RNA molecules. They are thus not only a generic feature of RNA sequence space but are relevant for the molecular evolution of biological RNA.     

Mutational Robustness Accelerates the Origin of Novel RNA Phenotypes through Phenotypic Plasticity

Novel phenotypes can originate either through mutations in existing genotypes or through phenotypic plasticity, the ability of one genotype to form multiple phenotypes. From molecules to organisms, plasticity is a ubiquitous feature of life, and a potential source of exaptations, adaptive traits that originated for nonadaptive reasons. Another ubiquitous feature is robustness to mutations, although it is unknown whether such robustness helps or hinders the origin of new phenotypes through plasticity. RNA is ideal to address this question, because it shows extensive plasticity in its secondary structure phenotypes, a consequence of their continual folding and unfolding, and these phenotypes have important biological functions. Moreover, RNA is to some extent robust to mutations. This robustness structures RNA genotype space into myriad connected networks of genotypes with the same phenotype, and it influences the dynamics of evolving populations on a genotype network. In this study I show that both effects help accelerate the exploration of novel phenotypes through plasticity. My observations are based on many RNA molecules sampled at random from RNA sequence space, and on 30 biological RNA molecules. They are thus not only a generic feature of RNA sequence space but are relevant for the molecular evolution of biological RNA.     

How a Replication Origin and Matrix Attachment Region Accelerate Gene Amplification under Replication Stress in Mammalian Cells

The gene amplification plays a critical role in the malignant transformation of mammalian cells. The most widespread method for amplifying a target gene in cell culture is the use of methotrexate (Mtx) treatment to amplify dihydrofolate reductase (Dhfr). Whereas, we found that a plasmid bearing both a mammalian origin of replication (initiation region; IR) and a matrix attachment region (MAR) was spontaneously amplified in mammalian cells. In this study, we attempted to uncover the underlying mechanism by which the IR/MAR sequence might accelerate Mtx induced Dhfr amplification. The plasmid containing the IR/MAR was extrachromosomally amplified, and then integrated at multiple chromosomal locations within individual cells, increasing the likelihood that the plasmid might be inserted into a chromosomal environment that permits high expression and further amplification. Efficient amplification of this plasmid alleviated the genotoxicity of Mtx. Clone-based cytogenetic and sequence analysis revealed that the plasmid was amplified in a chromosomal context by breakage-fusion-bridge cycles operating either at the plasmid repeat or at the flanking fragile site activated by Mtx. This mechanism explains how a circular molecule bearing IR/MAR sequences of chromosomal origin might be amplified under replication stress, and also provides insight into gene amplification in human cancer.     

Stepwise Evolution of the Herpes Simplex Virus Origin Binding Protein and Origin of Replication

The herpes simplex virus replicon consists of cis-acting sequences, oriS and oriL, and the origin binding protein (OBP) encoded by the UL9 gene. Here we identify essential structural features in the initiator protein OBP and the replicator sequence oriS, and we relate the appearance of these motifs to the evolutionary history of the alphaherpesvirus replicon. Our results reveal two conserved sequence elements in herpes simplex virus type 1, OBP; the RVKNL motif, common to and specific for all alphaherpesviruses, is required for DNA binding, and the WP XXXGAXXFXX L motif, found in a subset of alphaherpesviruses, is required for specific binding to the single strand DNA-binding protein ICP8. A 121-amino acid minimal DNA binding domain containing conserved residues is not soluble and does not bind DNA. Additional sequences present 220 amino acids upstream from the RVKNL motif are needed for solubility and function. We also examine the binding sites for OBP in origins of DNA replication and how they are arranged. NMR and DNA melting experiments demonstrate that origin sequences derived from many, but not all, alphaherpesviruses can adopt stable boxI/boxIII hairpin conformations. Our results reveal a stepwise evolutionary history of the herpes simplex virus replicon and suggest that replicon divergence contributed to the formation of major branches of the herpesvirus family.Herpesviruses have been found in animal species ranging from molluscs to man. According to the International Committee on Taxonomy of Viruses, the order of Herpesvirales consists of three families as follows: Alloherpesviridae, Herpesviridae, and Malacoherpesviridae (1). The subfamilies Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae are found within the family of Herpesviridae. The events leading to establishment of a new virus species are poorly understood, but in the case of herpesviruses it is commonly assumed that viruses co-evolve with their hosts (2). Herpesviruses have thus become well adapted to their hosts and may reside in a latent state in the host for a lifetime with little or no overt signs of infection. Upon reactivation, the infectious virus will be released. The viruses remain faithful to their hosts, and infections across species borders are rare but may under specific circumstances give rise to fatal disease.Replication of herpes simplex virus type 1 (HSV-1),² requires a cis-acting DNA sequence, the replicator, termed oriS or oriL, an initiator protein, OBP or UL9 protein, and a replisome composed of DNA polymerase, helicase-primase, and a single strand DNA-binding protein referred to as ICP8 or UL29 protein (3). OBP assisted by ICP8 can in an ATP-dependent reaction unwind double-stranded oriS (4, 5). The resulting single-stranded DNA adopts a hairpin conformation, which is stably bound by OBP (6, 7). The herpesvirus replisome once assembled on DNA is capable of synthesizing leading and lagging strands processively in a coordinated fashion (8). DNA replication is likely to start on circular molecules produced by the action of DNA ligase IV/XRCC4 and proceed in a θ-type manner (9, 10). Later, a rolling circle mode of replication dominates giving rise to characteristic head-to-tail concatemers. The initiator protein OBP appears to be strictly required only during the first few hours of the infectious cycle (11, 12).The HSV-1 origin binding protein was first isolated using an assay monitoring specific binding to HSV-1 oriS (13). A minimal DNA-binding site was identified using footprinting techniques as well as binding studies with double-stranded oligonucleotides (14). For alphaherpesviruses the high affinity binding site is always TTCGCAC, with a minor exception for CHV1 also referred to as monkey B virus (Table 1).3 The C-terminal 317 amino acids of alphaherpesvirus OBP can be isolated as a soluble protein, which remains capable of high affinity binding to the sequence TTCGCAC (15). The C-terminal domain of HSV-1 OBP, here referred to as ΔOBP, binds as a monomer to the major groove in the DNA and makes contacts with base pairs as well as the deoxyribose-phosphate backbone (16, 17). ΔOBP binds DNA specifically with an estimated K_d of 0.3 nm, a value that is highly influenced by the composition of the assay buffer (16). At high protein concentrations ΔOBP form aggregates, which, still in a sequence-specific manner, binds DNA (16). A number of studies have attempted to define amino acids involved in DNA binding (18–21). In addition, sequence comparisons between alphaherpesviruses and roseoloviruses have helped to identify amino acids in OBP potentially involved in DNA binding as well as corresponding recognition sequences in origins of DNA replication (22–25). However, a comprehensive and quantitative study of evolutionarily conserved amino acids required for DNA binding is still lacking.

TABLE 1

Functionally significant sequence motifs for herpes simplex virus replicon evolutionOrigins of DNA replication have been identified from sequence analysis. For roseoloviruses the sequences for two slightly different binding sites for OBP have been listed (31). The number and orientation of OBP-binding sites in relation to an AT-rich spacer sequence are schematically presented by symbols > and <. Note that since a virus often has more than one origin of replication they may exist as variants. This is indicated by symbols within parentheses. The conserved amino acids within the ICP8-binding motif are shown in boldface type. Sources and nomenclature for DNA sequences have been presented in Footnote 3.Open in a separate windowThe single strand DNA-binding protein, ICP8 encoded by the UL29 gene, is involved in initiation of DNA replication, and it also participates in the elongation phase at the replication fork (4, 5, 26). ICP8 forms a specific complex with OBP (26, 27). Studies with deletion mutants have demonstrated that important sequences are found close to the C terminus of OBP, but amino acids directly participating in the high affinity interaction have not been identified. The interaction is biologically significant, because deletion of the extreme C terminus enhances the helicase activity of OBP but reduces origin-dependent DNA synthesis (26).The HSV-1 oriS contains three copies of the binding site for OBP; two binding sites, box I and box II, are high affinity sites, and the third binding site, box III, has a very low affinity for OBP (14) (Fig. 1). All sites are required for efficient replication, and in a competitive situation there is a strong selection for the most efficient replicator sequence (28). Box III and box I are arranged in a palindrome, which becomes rearranged upon activation to form an alternative conformation, most likely a hairpin (4, 6, 7). Point mutations that prevent formation of a hairpin reduce replication, and compensatory mutations restore complementary base pairing as well as the ability to replicate (7).Open in a separate windowFIGURE 1.Schematic presentation of the herpes simplex virus replicon. Upper part, HSV-1 oriS. The linear genome contains three homologous replication origins, two copies of oriS and one copy oriL, and encodes seven replication proteins. Middle part, HSV-1 OBP. OBP or UL9 protein is a superfamily II DNA helicase as well as a sequence-specific DNA-binding protein. Here the helicase domain is represented by two connected ellipsoids, and the C-terminal DNA binding is drawn as a circle. The OBP-binding sites in oriS are shown. The OBP dimer binds two double-stranded DNA box I oligonucleotides but only one hairpin with a single-stranded tail (48). The figure is intended to demonstrate conformational changes affecting the DNA binding domain, referred to as ΔOBP, during activation of oriS. Lower part, DNA binding domain ΔOBP. A schematic presentation of the following three motifs discussed in this publication: the F553 XX KYL motif required for proper folding of the DNA binding domain, the R756VKNL motif necessary for DNA binding, and the W839PXXXGAXXFXXL motif involved in binding to ICP8.To learn more about the mechanism of virus evolution, we have examined the evolutionary history of some functionally significant features of the HSV-1 replicon and related them to a sequence-based evolutionary tree. The results indicate that replicon divergence, characterized by the stepwise appearance of the DNA-binding RVKNL motif, the WPXXXGAXXFXXL ICP8-binding motif, and the boxIII–boxI palindrome, may have played important roles in establishing major branches of the alphaherpesvirus tree.     

Modulating pyridoxamine-mediated transamination through a betabeta alpha motif peptide scaffold.

A pyridoxamine coenzyme amino acid chimera (Pam) was incorporated into a designed betabeta alpha motif peptide to explore the ability of a small synthetic peptide scaffold to influence coenzyme mediated transamination. Structural characterization of this peptide by CD and NMR spectroscopy suggested that the pyridoxamine containing residue was accommodated into the sheet region of the motif without gross structural perturbations. To investigate the ability of the peptide architecture to influence the amount and distribution of transamination product in the conversion of pyruvic acid to alanine, a family of 18 related peptides, CBP01-CBP18, was rapidly synthesized and purified in parallel. These peptides were designed to generate different peptide environments for the pyridoxamine functionality within the context of the structured betabeta alpha peptide motif. Studies of peptide-mediated transamination revealed clear trends in stereospecific production of L-alanine as a function of substitutions at positions five and seven of the motif. Furthermore, new trends favoring the other enantiomeric product resulted from the addition of copper(II) ion, a known chelator of the transamination reaction intermediates. In the presence of copper(II) ion the amount of alanine product generated was increased by up to 32-fold relative to a pyridoxamine model compound in the presence of copper(II) ion. These functional results, accompanied by further CD and NMR spectroscopic analysis of CBP14, one of the CBP family of peptides, suggest that small synthetic betabeta alpha motif peptides can be used to influence the functional properties of coenzymes.     

Universal Temporal Profile of Replication Origin Activation in Eukaryotes

Although replication proteins are conserved among eukaryotes, the sequence requirements for replication initiation differ between species. In all species, however, replication origins fire asynchronously throughout S phase. The temporal program of origin firing is reproducible in cell populations but largely probabilistic at the single-cell level. The mechanisms and the significance of this program are unclear. Replication timing has been correlated with gene activity in metazoans but not in yeast. One potential role for a temporal regulation of origin firing is to minimize fluctuations in replication end time and avoid persistence of unreplicated DNA in mitosis. Here, we have extracted the population-averaged temporal profiles of replication initiation rates for S. cerevisiae, S. pombe, D. melanogaster, X. laevis and H. sapiens from genome-wide replication timing and DNA combing data. All the profiles have a strikingly similar shape, increasing during the first half of S phase then decreasing before its end. A previously proposed minimal model of stochastic initiation modulated by accumulation of a recyclable, limiting replication-fork factor and fork-promoted initiation of new origins, quantitatively described the observed profiles without requiring new implementations.The selective pressure for timely completion of genome replication and optimal usage of replication proteins that must be imported into the cell nucleus can explain the generic shape of the profiles. We have identified a universal behavior of eukaryotic replication initiation that transcends the mechanisms of origin specification. The population-averaged efficiency of replication origin usage changes during S phase in a strikingly similar manner in a highly diverse set of eukaryotes. The quantitative model previously proposed for origin activation in X. laevis can be generalized to explain this evolutionary conservation.     

Replication Origin Selection Regulates the Distribution of Meiotic Recombination

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Modulating the strength of tetrel bonding through beryllium bonding

Quantum chemical calculations were performed to investigate the stability of the ternary complexes BeH₂···XMH₃···NH₃ (X?=?F, Cl, and Br; M?=?C, Si, and Ge) and the corresponding binary complexes at the atomic level. Our results reveal that the stability of the XMH₃···BeH₂ complexes is mainly due to both a strong beryllium bond and a weak tetrel–hydride interaction, while the XMH₃···NH₃ complexes are stabilized by a tetrel bond. The beryllium bond with a halogen atom as the electron donor has many features in common with a beryllium bond with an O or N atom as the electron donor, although they do exhibit some different characteristics. The stability of the XMH₃···NH₃ complex is dominated by the electrostatic interaction, while the orbital interaction also makes an important contribution. Interestingly, as the identities of the X and M atoms are varied, the strength of the tetrel bond fluctuates in an irregular manner, which can explained by changes in electrostatic potentials and orbital interactions. In the ternary systems, both the beryllium bond and the tetrel bond are enhanced, which is mainly ascribed to increased electrostatic potentials on the corresponding atoms and charge transfer. In particular, when compared to the strengths of the tetrel and beryllium bonds in the binary systems, in the ternary systems the tetrel bond is enhanced to a greater degree than the beryllium bond.

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Graphical Abstract A tetrel bond can be strengthened greatly by a beryllium bond

     

Origin and Direction of Haemophilus Bacteriophage HP1 DNA Replication

Rapidly growing Haemophilus influenzae strain Rd bacteria were infected with bacteriophage HP1 and DNA extracts prepared at various times thereafter. A number of phage genes scattered along the entire phage genome were quantitatively assayed by transformation. The kinetics of activity increases of these genes suggests that phage HP1 DNA replication begins at a fixed origin about one-quarter from the right end and that it proceeds to the left.     

Origin and Direction of Simian Virus 40 Deoxyribonucleic Acid Replication

Double-branched, circular, replicating deoxyribonucleic acid (DNA) molecules of simian virus 40 (SV40) have been cleaved by the R(1) restriction endonuclease from Escherichia coli. This enzyme introduces one double-strand break in SV40 DNA, at a specific site. The site of cleavage in the replicating molecules was used in this study to position the origin and the two branch points. Radioactively labeled molecules fractionated according to their extent of replication were evaluated after cleavage by sedimentation analysis and electron microscopy. The results demonstrate that the R(1) cleavage site is 33% of the genome length from the origin of replication and that both branch points are growing points. These data indicate that SV40 DNA replication is bidirectional and confirm other reports which have shown a unique origin of replication.     

Adaptive Immunity Restricts Replication of Novel Murine Astroviruses

The mechanisms of astrovirus pathogenesis are largely unknown, in part due to a lack of a small-animal model of disease. Using shotgun sequencing and a custom analysis pipeline, we identified two novel astroviruses capable of infecting research mice, murine astrovirus (MuAstV) STL1 and STL2. Subsequent analysis revealed the presence of at least two additional viruses (MuAstV STL3 and STL4), suggestive of a diverse population of murine astroviruses in research mice. Complete genomic characterization and subsequent phylogenetic analysis showed that MuAstV STL1 to STL4 are members of the mamastrovirus genus and are likely members of a new mamastrovirus genogroup. Using Rag1^−/− mice deficient in B and T cells, we demonstrate that adaptive immunity is required to control MuAstV infection. Furthermore, using Stat1^−/− mice deficient in innate signaling, we demonstrate a role for the innate immune response in the control of MuAstV replication. Our results demonstrate that MuAstV STL permits the study of the mechanisms of astrovirus infection and host-pathogen interactions in a genetically manipulable small-animal model. Finally, we detected MuAstV in commercially available mice, suggesting that these viruses may be present in academic and commercial research mouse facilities, with possible implications for interpretation of data generated in current mouse models of disease.     

Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design

Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC₅₀ of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds.     

Identification of the Rhesus Macaque Rhadinovirus Lytic Origin of DNA Replication

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.     

Location of the Origin of DNA Replication in Adenovirus Type 2

Utilizing the isolated left and right halves of both adenovirus type 2 and the nondefective adenovirus simian virus 40 hybrid (Ad2(+)ND(1)), studies were undertaken to find the site on the DNA molecules at which replication begins. The data are consistent with several models which include an initiation event at both ends and bidirectional growth.