Pisum sativum endonuclease. Studies on substrate specificity and possible use as a biochemical tool.
An endonuclease purified from germinating pea (Pisum sativum) seeds has been shown to catalyze the hydrolysis of heat-denatured single-stranded DNA. Since P. sativum endonuclease shows appreciable activity in the presence of DNA destabilizing agents and, unlike many similar endonucleases, significant activity at neutral pH, it is a potentially valuable tool for studies of the secondary structure of nucleic acids. The residual hydrolysis of duplex DNA is directed towards partially denatured, A,T-rich areas in native DNA. The rate of hydrolysis of deoxypolynucleotides was in the order poly(dT) greater than denatured DNA greater than poly(dA) greater than poly(dA-dT) = native DNA. Neither poly(dC), poly(dG) nor poly(dC).poly(dG) were attacked by the enzyme. Supercoiled, covalently closed circular phage PM2 form I DNA is converted to singly hit nicked circular form II and doubly hit linear from III duplexes. Prolonged treatment with enzyme does not further cleave the linear form III DNA. Addition of increasing concentrations of NaCl in the incubation mixture suppresses the conversion of form I to form II, but not the conversion of form II to form III, which is enhanced with the increasing ionic strength. The enzymatically relaxed circular form, I degree, obtained by unwinding of supercoiled DNA with a DNA-relaxing protein, is resistant to the action of the enzyme. Molecules with intermediate superhelix densities do not serve as substrates. The sites of cleavage of P. sativum endonuclease in PM2 DNA occur within regions that are readily denaturable in a topologically constrained superhelical molecule.
D000818:Animals; D002417:Cattle; D004247:DNA; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial; D004279:DNA, Viral; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity; D013950:Thymus Gland
D004269:DNA, Bacterial; D004279:DNA, Viral; D004247:DNA; D004262:DNA Restriction Enzymes