We have characterized a novel nuclease from the Kamchatka crab, designated duplex-specific nuclease (DSN). DSN displays a strong preference for cleaving double-stranded DNA and DNA in DNA-RNA hybrid duplexes, compared to single-stranded DNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is essentially higher than that for nonperfectly matched duplexes of the same length. Thus, DSN differentiates between one-nucleotide variations in DNA. We developed a novel assay for single nucleotide polymorphism (SNP) detection based on this unique property, termed "duplex-specific nuclease preference" (DSNP). In this innovative assay, the DNA region containing the SNP site is amplified and the PCR product mixed with signal probes (FRET-labeled short sequence-specific oligonucleotides) and DSN. During incubation, only perfectly matched duplexes between the DNA template and signal probe are cleaved by DSN to generate sequence-specific fluorescence. The use of FRET-labeled signal probes coupled with the specificity of DSN presents a simple and efficient method for detecting SNPs. We have employed the DSNP assay for the typing of SNPs in methyltetrahydrofolate reductase, prothrombin and p53 genes on homozygous and heterozygous genomic DNA.

Skin can age in two ways: (i) intrinsic or chronologic aging, which is the process of senescence that affects all body organs; and (ii) extrinsic aging which occurs as a consequence of exposure to environmental factors including sunlight and cigarette smoke. 1)The most important of these factors is sunlight, particularly exposure to ultraviolet (UV)B irradiation, which causes photoaging. The photoaging process increases skin fragility, laxity, blister formation, leathery appearance and formation of wrinkles. 2)UVB induces the production of matrix metalloproteinases (MMPs) by activating cellular signaling transduction pathways 3) ; MMPs are responsible for the degradation or synthesis inhibition of collagenous extracellular matrix in connective tissues.3) Collagen represents the main component of the extracellular matrix of dermal connective tissue, and its concentration decreases in chronoaging and photoaging.All matrix macromolecules in the dermis can be digested by MMPs acting alone or in combination. The MMPs form a family of structurally and functionally related zinc endopeptidases that exhibit various substrate specificities. 4) Once collagen is initially cleaved by MMP-1, MMP-3 and other MMPs, collagen breakdown is further promoted. The enzyme mainly responsible for collagen breakdown in skin is, MMP-1 (fibroblast collagenase), which cleaves type I, III, VII, VIII and X collagen.Human fibroblast collagenase was the first vertebrate collagenase both purified to homogeneity as a protein, and cloned as a cDNA. This enzyme has been designated as matrix metalloproteinase-1 (MMP-1) and has served as the prototype for all the interstitial collagenases. MMP-1 is also known as collagenase-1. Starting from the N terminus the following features of domain organization are observed. 5)The pre-domain specifies a signal rich in hydrophobic amino acids that destines the synthesized polypeptide to the endoplasmic reticulum where it is removed during the transportation of the molecule from the cell to the outside. The propeptide domain indicates a sequence that is responsible of keeping the pro-form inactive. It presents a cysteine residue 73 that is located in a conserved sequence PRCGVPD opposite to a zinc atom at the active-form site and coordinated to it through an -SH group. The enzymatic activity of the proform enzyme is turned on by the displacement of this cysteine residue ("cysteine switch") and occurs by proteolytic cleavage, or by chemical disruption as in the case of oxidation or treatment with mercurial compounds. 6)Recent studies have shown that hairless mice exposed to UVB developed wrinkled skin and significantly enhanced MMP-1 mRNA expression. However, they have shown that the inihibition of MMP-1 activities by a specific MMP inhibitor suppresses UVB-induced wrinkle formation.7) This evidence suggests that MMP-1 plays a major role in the process of photoaging.Brown seaweed has been a staple of both Korean and Japanese diets and has also been documented as being used in traditional Chinese medicine for over ...

HIV-1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. In contrast to canonical proteases (trypsin, chymotrypsin and proteinase K), IgGs and IgMs isolated from HIV-infected patients by affinity chromatography on immobilized IN specifically hydrolyzed only IN but not many other tested intact globular proteins. The sites of IN cleavage determined by MALDI mass spectrometry were localized mainly within seven known immunodominant regions of IN. Thin layer chromatography analysis has shown that the abzymes (Abzs) could also cleave 17 to 22-mer oligopeptides (OPs) corresponding to the immunodominant regions of IN sequence with a much higher rate than non-specific long peptides or three- and tetrapeptides of various sequence. Therefore, a prolonged incubation of IN with AIDS IgGs and IgMs having high catalytic activity usually produces many OPs of different length. Since anti-IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of the Abzs in counteracting the infection is possible.

BackgroundNucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking.ResultsUsing degenerative primers and the rapid amplification of cDNA ends (RACE) procedure, we cloned the Duplex-Specific Nuclease (DSN) gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5) and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact.ConclusionWe describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate specificity of DSN should prove valuable in certain molecular biology applications.

The psychrophilic marine bacterium, Cobetia marina, recovered from the mantle tissue of the marine mussel, Crenomytilus grayanus, which contained a gene encoding alkaline phosphatase (AP) with apparent biotechnology advantages. The enzyme was found to be more efficient than its counterparts and showed k cat value 10-to 100-fold higher than those of all known commercial APs. The enzyme did not require the presence of exogenous divalent cations and dimeric state of its molecule for activity. The recombinant enzyme (CmAP) production and purification were optimized with a final recovery of 2 mg of the homogenous protein from 1 L of the transgenic Escherichia coli Rosetta(DE3)/Pho40 cells culture. CmAP displayed a half-life of 16 min at 45°C and 27 min at 40°C in the presence of 2 mM EDTA, thus suggesting its relative thermostability in comparison with the known cold-adapted analogues. A high concentration of EDTA in the incubation mixture did not appreciably inhibit CmAP. The enzyme was stable in a wide range of pH (6.0-11.0). CmAP exhibited its highest activity at the reaction temperature of 40-50°C and pH 9.5-10.3. The structural features of CmAP could be the reason for the increase in its stability and catalytic turnover. We have modeled the CmAP 3D structure on the base of the high-quality experimental structure of the close homologue Vibrio sp. AP (VAP) and mutated essential residues predicted to break Mg 2+ bonds in CmAP. It seems probable that the intrinsically tight binding of catalytic and structural metal ions together with the flexibility of intermolecular and intramolecular links in CmAP could be attributed to the adapted mutualistic lifestyle in oceanic waters.