Enzymes BamHI Research Articles

Genotyping of the Pseudorabies Virus by Multiplex PCR Followed by Restriction Enzyme Analysis

Suid herpesvirus 1 (SuHV-1) is the causative agent of Aujeszky's disease. The infectious agent has only one serotype, but it was classified by restriction enzyme analysis of the whole genome into four genotypes, named I to IV. The aim of this study was to standardize a rapid method for genotyping SuHV-1 without virus isolation, using a multiplex-PCR followed by enzymatic restriction analysis. The complete genome of the virus was analyzed in silico to determine the restriction sites for the enzyme BamHI. Primers were designed to flank sites with emphasis on certain points of differentiation of genotypes. The standard PCRs were able to detect the SuHV-1 and also to differentiate genotypes from brain tissue of infected pigs. The BamHI-PCR is a rapid, practical, and sensitive way to genotype SuHV-1.

A new dispersed element in the genome of the catfish Hisonotus leucofrenatus (Teleostei

Eight restriction enzymes were used in the prospection and isolation of repetitive sequences in the genome of the catfish Hisonotus leucofrenatus, a siluriform fish species that presents a large heterochromatic block in the W chromosome. In the tested enzymes, only the BamHI enzyme revealed a distinct band of 224 bp G+C value of 37%. In comparative analyses with sequences already available in the GenBank, the BamHI fragment sequence showed similarity with part of a Lepidoptera transposon. Fluorescent in situ hybridization (FISH) revealed that this sequence presents a dispersed pattern in the genome of H. leucofrenatus, forming clusters in some chromosome pairs in the pericentromeric region, which frequently are rich in constitutive heterochromatin. Based on the analysis performed, it can be inferred that the HLBam fragment constitutes a genomically dispersed transposon type element. It can be considered that the findings in this study can contribute to a better understanding of the organization and distribution of transposable elements in the genome of teleost fish.

A novel vector for a rapid generation of fiber-mutant adenovirus based on one step ligation and quick screening of positive clones

The generation of fiber-modified adenoviral vector has proven difficult. In the paper, we developed a new system for rapid construction of fiber-modified adenoviral vector containing foreign peptides in the HI loop or C-terminal of the fiber knob. The new system was established through the following processes. First, a unique BamHI mutation was made in the genome of Ad5 without causing amino acid change. Second, two unique restriction enzymes BamHI and SfuI, both with sticky end, were introduced in the HI loop or C-terminal of Ad5 fiber knob. Third, a lacza expression cassette was placed between BamHI and SfuI sites for a quick identification of positive cloning based on white–blue color screening. This system allows generation of recombinant adenoviral vector by a single step, in vitro ligation followed by quick white–color positive clone screening. To prove the principle of the method, Ad5HI-RGD by modifying HI-loop of the fiber knob with RGD motif and Ad5Cter-PK7 by modifying C-terminal of the knob with poly-lysine (pK7) were successfully generated in vitro. Ad5 with a knob modified in the HI loop of the fiber with Tat-PTD, NGR or SIKVAV peptide were also successfully developed. The transduction of the modified viruses for Hela, U87 MG and MDA-MB-231 cells was investigated in vitro compared with unmodified Ad5. In conclusion, the new vector system allows for a rapid generation of fiber-mutant adenovirus and provides useful tool for gene function analysis and cancer gene therapy.

Inhibition of Restriction Enzymes EcoRI, BamHI and HindIII by Phenethylphenylphthalimides Derived from Thalidomide

We discovered inhibitors of the restriction enzymes EcoRI, BamHI and HindIII by screening our library of compounds with a phenethylphenylphthalimide skeleton, based on α-glucosidase inhibitors and liver X receptor antagonists derived from thalidomide. Structural development afforded the potent restriction enzyme inhibitors 25 and 26.

Homophilic Adhesion Mechanism of Neurofascin, a Member of the L1 Family of Neural Cell Adhesion Molecules

The L1 family neural cell adhesion molecules play key roles in specifying the formation and remodeling of the neural network, but their homophilic interaction that mediates adhesion is not well understood. We report two crystal structures of a dimeric form of the headpiece of neurofascin, an L1 family member. The four N-terminal Ig-like domains of neurofascin form a horseshoe shape, akin to several other immunoglobulin superfamily cell adhesion molecules such as hemolin, axonin, and Dscam. The neurofascin dimer, captured in two crystal forms with independent packing patterns, reveals a pair of horseshoes in trans-synaptic adhesion mode. The adhesion interaction is mediated mostly by the second Ig-like domain, which features an intermolecular β-sheet formed by the joining of two individual GFC β-sheets and a large but loosely packed hydrophobic cluster. Mutagenesis combined with gel filtration assays suggested that the side chain hydrogen bonds at the intermolecular β-sheet are essential for the homophilic interaction and that the residues at the hydrophobic cluster play supplementary roles. Our structures reveal a conserved homophilic adhesion mode for the L1 family and also shed light on how the pathological mutations of L1 affect its structure and function.

Rapid targeting and isolation of the -like globin gene cluster fragment from AA, AS and SS genotypesusing BamHI restriction enzyme

The β-globin gene cluster contains the β-globin gene on which the substitution of the 17thnucleotide gives rise to sickle cell anemia and other β-globin gene variants with varying severity. Recent therapy measures focus on understanding the structure and mechanism of expression of the β-globin gene. In this study, we attempted to isolate a gene fragment containing the complete set of the human β-globin gene cluster from AA, AS and SS blood types, using the restriction enzyme BamHI which has a recognition site within the 19-kb 3’ cluster region downstream the β-globin gene cluster. DNA fragment size of 69.65 kb was generated by the enzyme from the genome of AS and SS blood types while the fragment generated from the AA blood type was 70.28 kb. Fragments generated using Bam HI was all comparable and close to the documented size of the β-globin gene cluster. In addition to the 69.65 kb fragment generated from the DNA from SS blood type, a novel 5.73 kb fragment was also visualized which was absent in the lanes containing restricted DNA from the AA blood type. Key words: BamHI, beta-globin, genotype, isolation.

Identification of the Tibetan fox (Vulpes ferrilata) and the red fox (Vulpes vulpes) by copro‐DNA diagnosis

Studies on feeding habits based on faeces dissecting are imperative to understand the natural history of Tibetan foxes and their functions in the transmission of a lethal zoonotic parasite, Echinococcus multilocularis. However, Tibetan foxes and red foxes live sympatrically on the Tibetan plateau, China. Therefore, the faeces of Tibetan foxes must be distinguished from those of red foxes. We established a diagnostic method to distinguish the faeces of the two species by amplifying a portion of the mitochondrial cytochrome b gene (cytb) and digesting with the restriction enzymes BamHI and SspI, to produce specific diagnostic banding patterns. This PCR-RFLP assay enabled rapid, accurate and easily performed identification and differentiation of the two species.

Lon Protease Quality Control of Presecretory Proteins in Escherichia coli and Its Dependence on the SecB and DnaJ (Hsp40) Chaperones

Various environmental insults result in irreversible damage to proteins and protein complexes. To cope, cells have evolved dedicated protein quality control mechanisms involving molecular chaperones and proteases. Here, we provide both genetic and biochemical evidence that the Lon protease and the SecB and DnaJ/Hsp40 chaperones are involved in the quality control of presecretory proteins in Escherichia coli. We showed that mutations in the lon gene alleviate the cold-sensitive phenotype of a secB mutant. Such suppression was not observed with either clpP or clpQ protease mutants. In comparison to the respective single mutants, the double secB lon mutant strongly accumulates aggregates of SecB substrates at physiological temperatures, suggesting that the chaperone and the protease share substrates. These observations were extended in vitro by showing that the main substrates identified in secB lon aggregates, namely proOmpF and proOmpC, are highly sensitive to specific degradation by Lon. In contrast, both substrates are significantly protected from Lon degradation by SecB. Interestingly, the chaperone DnaJ by itself protects substrates better from Lon degradation than SecB or the complete DnaK/DnaJ/GrpE chaperone machinery. In agreement with this finding, a DnaJ mutant protein that does not functionally interact in vivo with DnaK efficiently suppresses the SecB cold-sensitive phenotype, highlighting the role of DnaJ in assisting presecretory proteins. Taken together, our data suggest that when the Sec secretion pathway is compromised, a pool of presecretory proteins is transiently maintained in a translocation-competent state and, thus, protected from Lon degradation by either the SecB or DnaJ chaperones.

Characterization of Single Molecule Protein Induced Fluorescence Enhancement (PIFE) as an Alternative to SmFRET

Forster resonance energy transfer (FRET) is a widely used technique for real time single molecule detection. FRET measurement is based on distance changes between donor and acceptor fluorophores, hence the requirement for fluorophore attachment of the molecules under study. Single fluorophore labeling of protein is often difficult to achieve, rendering poor efficiency and specificity of labeling. Furthermore, proteins with high Kd which requires addition of high protein concentration would make single molecule detection impossible even with properly labeled proteins. Recently, PIFE, an alternative fluorescence assay was developed for probing translocational movement of an antiviral protein, RIG-I (1). PIFE employs a single fluorophore which exhibits enhanced quantum yield when approached by a protein at a close proximity. Although this photophysical effect is correlated with the lifetime change of the corresponding fluorophore (2) the sensitivity and the distance range of the method needs to be further characterized.We performed a systematic study of a single molecule PIFE where we monitored binding of a restriction enzyme BamHI to Cy3 labeled DNA. Preliminary data shows that the sequence specific binding of BamHI to DNA in a buffer containing calcium results in an increase of Cy3 intensity. The overall level of fluorescence increase shows dependence on the protein concentration and single molecule traces display binding and unbinding as discrete steps of increase and decrease in Cy3 intensity respectively. We confirmed the sequence specific cleavage activity of BamHI triggered by the presence of magnesium.

Nucleic Acid Therapeutic Carriers with On-Demand Triggered Release

Biohybrid platforms such as synthetic polymer networks engineered from artificial and natural materials hold immense potential as drug and gene delivery vehicles. Here, we report the synthesis and characterization of novel polymer networks that release oligonucleotide sequences via enzymatic and physical triggers. Chemical monomers and acrylated oligonucleotides were copolymerized into networks, and phosphoimaging revealed that 70% of the oligonucleotides were incorporated into the networks. We observed that the immobilized oligonucleotides were readily cleaved when the networks were incubated with the type II restriction enzyme BamHI. The diffusion of the cleaved fragments through the macromolecular chains resulted in relatively constant release profiles very close to zero-order. To our knowledge, this is the first study which harnesses the sequence-specificity of restriction endonucleases as triggering agents for the cleavage and release of oligonucleotide sequences from a synthetic polymer network. The polymer networks exhibited an oligonucleotide diffusion coefficient of 5.6 x 10(-8) cm(2)/s and a diffusional exponent of 0.92. Sigmoidal temperature responsive characteristics of the networks matched the theoretical melting temperature of the oligonucleotides and indicated a cooperative melting transition of the oligonucleotides. The networks were also triggered to release a RNA-cleaving deoxyribozyme, which degraded a HIV-1 mRNA transcript in vitro. To tailor release profiles of the oligonucleotides, we controlled the structure of the macromolecular architecture of the networks by varying their cross-linking content. When incubated with DNase I, networks of cross-linking content 0.15%, 0.22%, and 0.45% exhibited oligonucleotide diffusion coefficients of 1.67 x 10(-8), 7.65 x 10(-9), and 2.7 x 10(-9) cm(2)/s, and diffusional exponents of 0.55, 0.8, and 0.8, respectively. The modular nature of our platform promises to open new avenues for the creation and optimization of a rich toolbox of novel drug and gene delivery platforms. We anticipate further inquiry into nucleic acid based programmable on-demand switches and modulatory devices of exquisite sensitivity and control.

First Report of the Occurrence of Sweet potato leaf curl virus in Tall Morningglory (Ipomoea purpurea) in China.

Natural occurrence of Sweet potato leaf curl virus (SPLCV) has been reported in Ipomoea batatas (sweet potato, Convolvulaceae) or I. indica (Convolvulaceae) in several countries including the United States, Sicily, and China (1-3). In September of 2007, while collecting samples showing begomovirus-like symptoms in the Chinese province of Fujian, we observed tall morningglory (I. purpurea (L.) Roth, also known as Pharbitis purpurea (L.) Voigt), plants with slightly yellow mosaic and crinkled leaves. Total DNA was extracted from leaves of these plants and tested by rolling circle amplification (4). Amplification products were digested by the restriction enzyme BamHI for 30 min. Restriction products (2.8 kb) were then cloned into pMD18T vector (Takara Biotechnology, China) and sequenced. Comparison of complete DNA sequences by Clustal V analysis revealed that these samples were infected by the same virus, and an isolate denoted F-p1 was selected for further sequence analysis. F-p1 was 2,828 nucleotides, with the typical genomic organization of begomoviral DNA-A (GenBank Accession No. FJ515896). F-p1 was compared with the DNA sequences available in the NCBI database using BLAST. The whole DNA sequence showed the highest nucleotide sequence identity (92.1%) with an isolate of SPLCV (GenBank Accession No. FJ176701) from Jiangsu Province of China. The result confirmed that the samples from the symptomatic tall morningglory were infected by SPLCV. To our knowledge, this is the first report of the natural occurrence of SPLCV in I. purpurea, a common weed species in China.

Major Urinary Protein-1 Increases Energy Expenditure and Improves Glucose Intolerance through Enhancing Mitochondrial Function in Skeletal Muscle of Diabetic Mice

Major urinary protein-1 (MUP-1) is a low molecular weight secreted protein produced predominantly from the liver. Structurally it belongs to the lipocalin family, which carries small hydrophobic ligands such as pheromones. However, the physiological functions of MUP-1 remain poorly understood. Here we provide evidence demonstrating that MUP-1 is an important player in regulating energy expenditure and metabolism in mice. Both microarray and real-time PCR analysis demonstrated that the MUP-1 mRNA abundance in the liver of db/db obese mice was reduced by approximately 30-fold compared with their lean littermates, whereas this change was partially reversed by treatment with the insulin-sensitizing drug rosiglitazone. In both dietary and genetic obese mice, the circulating concentrations of MUP-1 were markedly decreased compared with the lean controls. Chronic elevation of circulating MUP-1 in db/db mice, using an osmotic pump-based protein delivery system, increased energy expenditure and locomotor activity, raised core body temperature, and decreased glucose intolerance as well as insulin resistance. At the molecular level, MUP-1-mediated improvement in metabolic profiles was accompanied by increased expression of genes involved in mitochondrial biogenesis, elevated mitochondrial oxidative capacity, decreased triglyceride accumulation, and enhanced insulin-evoked Akt signaling in skeletal muscle but not in liver. Altogether, these findings raise the possibility that MUP-1 deficiency might contribute to the metabolic dysregulation in obese/diabetic mice, and suggest that the beneficial metabolic effects of MUP-1 are attributed in part to its ability in increasing mitochondrial function in skeletal muscle.

A Novel Protein Acts as a Negative Regulator of Prophenoloxidase Activation and Melanization in the Freshwater Crayfish Pacifastacus leniusculus

Melanization is an important immune component of the innate immune system of invertebrates and is vital for defense as well as for wound healing. In most invertebrates melanin synthesis is achieved by the prophenoloxidase-activating system, a proteolytic cascade similar to vertebrate complement. Even though melanin formation is necessary for host defense in crustaceans and insects, the process needs to be tightly regulated because of the hazard to the animal of unwanted production of quinone intermediates and melanization in places where it is not suitable. In the present study we have identified a new melanization inhibition protein (MIP) from the hemolymph of the crayfish, Pacifastacus leniusculus. Crayfish MIP has a similar function as the insect MIP molecule we recently discovered in the beetle Tenebrio molitor but interestingly has a completely different sequence. Crayfish MIP as well as Tenebrio MIP do not affect phenoloxidase activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin. Importantly, crayfish MIP in contrast to Tenebrio MIP contains a fibrinogen-like domain, most similar to the substrate recognition domain of vertebrate l-ficolins. Surprisingly, an Asp-rich region similar to that found in ficolins that is likely to be involved in Ca2+ binding is present in crayfish MIP. However, crayfish MIP did not show any hemagglutinating activity as is common for the vertebrate ficolins. A mutant form of MIP with a deletion lacking four Asp amino acids from the Asp-rich region lost most of its activity, implicating that this part of the protein is involved in regulating the prophenoloxidase activating cascade. Overall, a new negative regulator of melanization was identified in freshwater crayfish that shows interesting parallels with proteins (i.e. ficolins) involved in vertebrate immune response.

Calcium-dependent Binding of HCN1 Channel Protein to Hair Cell Stereociliary Tip Link Protein Protocadherin 15 CD3

The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform expressed in trout saccular hair cells, was found by yeast two-hybrid protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin 15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair cell stereocilia, consistent with gradated distribution expected for tip link sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries of rat cochlear inner and outer hair cells, and HCN1 protein was immunolocalized to cochlear hair cell stereocilia. As predicted by the trout hair cell model, the amino terminus of rat organ of Corti HCN1 was found by yeast two-hybrid analysis to bind the carboxyl terminus of protocadherin 15 CD3, a tip link protein implicated in mechanosensory transduction. Specific binding between HCN1 and protocadherin 15 CD3 was confirmed with pull-down assays and surface plasmon resonance analysis, both predicting dependence on Ca(2+). In the presence of calcium chelators, binding between HCN1 and protocadherin 15 CD3 was characterized by a K(D) = 2.39 x 10(-7) m. Ca(2+) at 26.5-68.0 microm promoted binding, with K(D) = 5.26 x 10(-8) m (at 61 microm Ca(2+)). Binding by deletion mutants of protocadherin 15 CD3 pointed to amino acids 158-179 (GenBank accession number XP_238200), with homology to the comparable region in trout hair cell protocadherin 15a-like protein, as necessary for binding to HCN1. Amino terminus binding of HCN1 to HCN1, hypothesized to underlie HCN1 channel formation, was also found to be Ca(2+)-dependent, although the binding was skewed toward a lower effective maximum [Ca(2+)] than for the HCN1 interaction with protocadherin 15 CD3. Competition may therefore exist in vivo between the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3 favored between 26.5 and 68 microm Ca(2+). Taken together, the evidence supports a role for HCN1 in mechanosensory transduction of inner ear hair cells.

KloningOpen Reading Frame (Orf) ESAT-6 (Early Secretory Antigenic Target-6) Mycobacterium Tuberculosis Ke Escherichia ColiBL21 (DE3)

Tuberculosis (TB) caused by Mycobacterium tuberculosis is an infectious diseases leading to significant death toll in most partof the world. Vaccination with BCG, a TB vaccine, is still a common practice until now. In general, the people in Indonesia receive BCG vaccine during their early childhood, but the efficacy of the vaccine would not last long to adulthood, which allow themto get potential latent TB infection. This latent TB infection might be detected by tuberculin skin test (TST), however, the weakness is that false positive reactions are commonly found due to cross reaction between antibodies produced during BCG vaccination and purified protein derivative (PPD). Alternatively, its detection could be performed by identifying immunodominant antigen to M. tuberculosis. The Early Secretory AntigenicTarget-6 (ESAT-6) for antibody based serological detection with high sensitivity and specificity could also be applied. The purpose of this study was to clone the open reading frame (orf) of ESAT-6 from Mycobacterium tuberculosis into Escherichia coli BL21 (DE3). In this method, orf ESAT-6 was ligated to the expression vector pET-32b and transformed into E. coli BL21 (DE3). Characterization of clones was carried out by cutting the recombinant plasmid using restriction enzymes BamH1 and XhoI. The result showed that three colonies with recombinant plasmid pET-32b-ESAT-6 were obtained. Sequencing of the DNA insert was then performed using the universal T7 primer. Characterization of white colonies with restriction enzymes showed two bands i.e. 288 bp and 5900 bp for orf ESAT-6 and pET32b vector respectively. BLAST analysis of sequence showed 100% homology.

Initiation of Mammalian O-Mannosylation in Vivo Is Independent of a Consensus Sequence and Controlled by Peptide Regions within and Upstream of the α-Dystroglycan Mucin Domain

To reveal insight into the initiation of mammalian O-mannosylation in vivo, recombinant glycosylation probes containing sections of human alpha-dystroglycan (hDG) were expressed in epithelial cell lines. We demonstrate that O-mannosylation within the mucin domain of hDG occurs preferentially at Thr/Ser residues that are flanked by basic amino acids. Protein O-mannosylation is independent of a consensus sequence, but strictly dependent on a peptide region located upstream of the mucin domain. This peptide region cannot be replaced by other N-terminal peptides, however, it is not sufficient to induce O-mannosylation on a structurally distinct mucin domain in hybrid constructs. The presented in vivo evidence for a more complex regulation of mammalian O-mannosylation contrasts with a recent in vitro study of O-mannosylation in human alpha-dystroglycan peptides indicating the existence of an 18-meric consensus sequence. We demonstrate in vivo that the entire region p377-417 is necessary and sufficient for O-mannosylation initiation of hDG, but not of MUC1 tandem repeats. The feature of a doubly controlled initiation process distinguishes mammalian O-mannosylation from other types of O-glycosylation, which are largely controlled by structural properties of the substrate positions and their local peptide environment.

A Kinetic Study of Human Protein Arginine N-Methyltransferase 6 Reveals a Distributive Mechanism

Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-L-methionine to arginine residues within proteins, forming S-adenosyl-L-homocysteine as well as omega-N(G)-monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) residues in the process. We have characterized the kinetic mechanism of recombinant His-tagged PRMT6 using a mass spectrometry method for monitoring the methylation of a series of peptides bearing a single arginine, MMA, or aDMA residue. We find that PRMT6 follows an ordered sequential mechanism in which S-adenosyl-L-methionine binds to the enzyme first and the methylated product is the first to dissociate. Furthermore, we find that the enzyme displays a preference for the monomethylated peptide substrate, exhibiting both lower K(m) and higher V(max) values than what are observed for the unmethylated peptide. This difference in substrate K(m) and V(max), as well as the lack of detectable aDMA-containing product from the unmethylated substrate, suggest a distributive rather than processive mechanism for multiple methylations of a single arginine residue. In addition, we speculate that the increased catalytic efficiency of PRMT6 for methylated substrates combined with lower K(m) values for native protein methyl acceptors may obscure this distributive mechanism to produce an apparently processive mechanism.

Variation in the miRNA-433 Binding Site of FGF20 Confers Risk for Parkinson Disease by Overexpression of α-Synuclein

Parkinson disease (PD) is a common neurodegenerative disorder caused by environmental and genetic factors. We have previously shown linkage of PD to chromosome 8p. Subsequently, fibroblast growth factor 20 (FGF20) at 8p21.3-22 was identified as a risk factor in several association studies. To identify the risk-conferring polymorphism in FGF20, we performed genetic and functional analysis of single-nucleotide polymorphisms within the gene. In a sample of 729 nuclear families with 1089 affected and 1165 unaffected individuals, the strongest evidence of association came from rs12720208 in the 3' untranslated region of FGF20. We show in several functional assays that the risk allele for rs12720208 disrupts a binding site for microRNA-433, increasing translation of FGF20 in vitro and in vivo. In a cell-based system and in PD brains, this increase in translation of FGF20 is correlated with increased alpha-synuclein expression, which has previously been shown to cause PD through both overexpression and point mutations. We suggest a novel mechanism of action for PD risk in which the modulation of the susceptibility gene's translation by common variations interfere with the regulation mechanisms of microRNA. We propose this is likely to be a common mechanism of genetic modulation of individual susceptibility to complex disease.

Regulation of Protein Arginine Methyltransferase 8 (PRMT8) Activity by Its N-terminal Domain

Human protein arginine methyltransferase PRMT8 has been recently described as a type I enzyme in brain that is localized to the plasma membrane by N-terminal myristoylation. The amino acid sequence of human PRMT8 is almost 80% identical to human PRMT1, the major protein arginine methyltransferase activity in mammalian cells. However, the activity of a recombinant PRMT8 GST fusion protein toward methyl-accepting substrates is much lower than that of a GST fusion of PRMT1. We show here that both His-tagged and GST fusion species lacking the initial 60 amino acid residues of PRMT8 have enhanced enzymatic activity, suggesting that the N-terminal domain may regulate PRMT8 activity. This conclusion is supported by limited proteolysis experiments showing an increase in the activity of the digested full-length protein, consistent with the loss of the N-terminal domain. In contrast, the activity of the N-terminal truncated protein was slightly diminished by limited proteolysis. Significantly, we detect automethylation at two sites in the N-terminal domain, as well as binding sites for SH3 domain-containing proteins. We suggest that the N-terminal domain may function as an autoregulator that may be displaced by interaction with one or more physiological inducers.

Hexameric ring structure of human MCM10 DNA replication factor

The DNA replication factor minichromosome maintenance 10 (MCM10) is a conserved, abundant nuclear protein crucial for origin firing. During the transition from pre-replicative complexes to pre-initiation complexes, MCM10 recruitment to replication origins is required to provide a physical link between the MCM2-7 complex DNA helicase and DNA polymerases. Here, we report the molecular structure of human MCM10 as determined by electron microscopy and single-particle analysis. The MCM10 molecule is a ring-shaped hexamer with large central and smaller lateral channels and a system of inner chambers. This structure, together with biochemical data, suggests that this important protein uses its architecture to provide a docking module for assembly of the molecular machinery required for eukaryotic DNA replication.