Identification, genetic analysis and characterization of a sugar-non-specific nuclease from the cyanobacterium Anabaena sp. PCC 7120.
A nuclease that could be recovered from the supernatant of cultures, as well as from cell-free extracts, of the cyanobacterium Anabaena sp. PCC 7120 was identified as a 29 kDa polypeptide by its ability to degrade DNA after electrophoresis in DNA-containing SDS-polyacrylamide gels. Some clones of a gene library of strain PCC 7120 established in Escherichia coli were found to produce the 29 kDa nuclease. The nucA gene encoding this nuclease was subcloned and sequenced. The deduced polypeptide, NucA, had a molecular weight of 29,650, presented a presumptive signal peptide in its N-terminal region and showed homology to the products of the nuc gene from Serratia marcescens and the NUC1 gene from Saccharomyces cerevisiae. The NucA protein from Anabaena itself, or from the cloned nucA gene expressed in E. coli, catalysed the degradation of both RNA and DNA, had the potential to act as an endonuclease, and functioned best in the presence of Mn2+ or Mg2+. An Anabaena nucA insertional mutant was generated which failed to produce the 29 kDa nuclease.
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Facultad de Biología, Spain.
A M
AM
Muro-Pastor
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E
Flores
A
A
Herrero
C P
CP
Wolk
D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003851:Deoxyribonucleases / Q000235:genetics; D004720:Endonucleases; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010727:Phosphoric Diester Hydrolases / Q000235:genetics* / Q000302:isolation & purification; D021382:Protein Sorting Signals; D012260:Ribonucleases / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D021382:Protein Sorting Signals; D003851:Deoxyribonucleases; D004720:Endonucleases; D012260:Ribonucleases; C085858:sugar-nonspecific nuclease; D010727:Phosphoric Diester Hydrolases