RESUMO
A single-strand-specific endonuclease from mung bean sprouts is widely used in molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.
Assuntos
Divisão Celular/fisiologia , DNA de Plantas/metabolismo , DNA de Cadeia Simples/metabolismo , Endodesoxirribonucleases/metabolismo , Fabaceae/enzimologia , Plantas Medicinais , Sequência de Bases , Clonagem Molecular , Reparo do DNA , Replicação do DNA , DNA de Plantas/biossíntese , DNA Super-Helicoidal/metabolismo , Dados de Sequência Molecular , Protamina Quinase/análise , Análise de Sequência de DNA , Distribuição TecidualRESUMO
The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its Km using synthetic S-allylcysteine sulfoxide (+ isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N-terminal of the intact polypeptide. The second step involved screening of garlic lambda gt11 and lambda ZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3' noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from alpha wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.