Purification, Characterization and CDNA Cloning of an Endo-exonuclease from the Basidiomycete Fungus Armillaria Mellea

Overview

Journal Biochem J

Specialty Biochemistry

Date 1999 Apr 24

PMID 10215611

Authors

V Healy

S Doonan

T V McCarthy

Affiliations

Soon will be listed here.

Abstract

We have purified an endo-exonuclease from the fruiting body of the basidiomycete fungus Armillaria mellea by using an ethanol fractionation step, followed by two rounds of column chromatography. The enzyme had an apparent molecular mass of 17500 Da and was shown to exist as a monomer by gel-filtration analysis. The nuclease was active on both double-stranded and single-stranded DNA but not on RNA. It was optimally active at pH8.5 and also exhibited a significant degree of thermostability. Three bivalent metal ions, Mg2+, Co2+ and Mn2+, acted as cofactors in the catalysis. It was also inhibited by high salt concentrations: activity was completely abolished at 150 mM NaCl. The nuclease possessed both endonuclease activity on supercoiled DNA and a 3'-5' (but not a 5'-3') exonuclease activity. It generated 5'-phosphomonoesters on its products that, after a prolonged incubation, were hydrolysed to a mixture of free mononucleotides and small oligonucleotides ranging in size from two to eight bases. Elucidation of its N-terminal amino acid sequence permitted the cDNA cloning of the A. mellea nuclease via a PCR-based approach. Peptide mapping of the purified enzyme generated patterns consistent with the amino acid sequence coded for by the cloned cDNA. A BLAST search of the SwissProt database revealed that A. mellea nuclease shared significant amino acid similarity with two nucleases from Bacillus subtilis, suggesting that the three might constitute a distinct class of nucleolytic enzymes.

References

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Fraser M, Koa H, Chow T . Neurospora endo-exonuclease is immunochemically related to the recC gene product of Escherichia coli. J Bacteriol. 1990; 172(1):507-10. PMC: 208465. DOI: 10.1128/jb.172.1.507-510.1990. View

Lu B, Sakaguchi K . An endo-exonuclease from meiotic tissues of the basidiomycete Coprinus cinereus. Its purification and characterization. J Biol Chem. 1991; 266(31):21060-6. View

Zassenhaus H, Hofmann T, Uthayashanker R, Vincent R, Zona M . Construction of a yeast mutant lacking the mitochondrial nuclease. Nucleic Acids Res. 1988; 16(8):3283-96. PMC: 336494. DOI: 10.1093/nar/16.8.3283. View

Chow T, Resnick M . An endo-exonuclease activity of yeast that requires a functional RAD52 gene. Mol Gen Genet. 1988; 211(1):41-8. DOI: 10.1007/BF00338391. View

ROSENTHAL A, Lacks S . Nuclease detection in SDS-polyacrylamide gel electrophoresis. Anal Biochem. 1977; 80(1):76-90. DOI: 10.1016/0003-2697(77)90627-3. View

van Sinderen D, Kiewiet R, Venema G . Differential expression of two closely related deoxyribonuclease genes, nucA and nucB, in Bacillus subtilis. Mol Microbiol. 1995; 15(2):213-23. DOI: 10.1111/j.1365-2958.1995.tb02236.x. View

Smith H, Marley G . Purification and properties of HindII and HindIII endonucleases from Haemophilus influenzae Rd. Methods Enzymol. 1980; 65(1):104-8. DOI: 10.1016/s0076-6879(80)65015-0. View

Hildebrandt E, Boykin D, Kumar A, Tidwell R, Dykstra C . Identification and characterization of an endo/exonuclease in Pneumocystis carinii that is inhibited by dicationic diarylfurans with efficacy against Pneumocystis pneumonia. J Eukaryot Microbiol. 1998; 45(1):112-21. DOI: 10.1111/j.1550-7408.1998.tb05078.x. View

10.

Reyes F, Villanueva P, Alfonso C . Nucleases in the autolysis of filamentous fungi. FEMS Microbiol Lett. 1990; 57(1-2):67-72. DOI: 10.1016/0378-1097(90)90414-l. View

11.

Meselson M, Radding C . A general model for genetic recombination. Proc Natl Acad Sci U S A. 1975; 72(1):358-61. PMC: 432304. DOI: 10.1073/pnas.72.1.358. View

12.

Altschul S, Gish W, Miller W, Myers E, Lipman D . Basic local alignment search tool. J Mol Biol. 1990; 215(3):403-10. DOI: 10.1016/S0022-2836(05)80360-2. View

13.

Fraser M, Tynan S, Papaioannou A, Ireland C, Pittman S . Endo-exonuclease of human leukaemic cells: evidence for a role in apoptosis. J Cell Sci. 1996; 109 ( Pt 9):2343-60. DOI: 10.1242/jcs.109.9.2343. View

14.

Friedhoff P, Kolmes B, Gimadutdinow O, Wende W, Krause K, Pingoud A . Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis. Nucleic Acids Res. 1996; 24(14):2632-9. PMC: 146012. DOI: 10.1093/nar/24.14.2632. View

15.

Szostak J, Rothstein R, Stahl F . The double-strand-break repair model for recombination. Cell. 1983; 33(1):25-35. DOI: 10.1016/0092-8674(83)90331-8. View

16.

Fraser M . Endo-exonucleases: enzymes involved in DNA repair and cell death?. Bioessays. 1994; 16(10):761-6. DOI: 10.1002/bies.950161011. View

17.

Kolodkin A, Klar A, Stahl F . Double-strand breaks can initiate meiotic recombination in S. cerevisiae. Cell. 1986; 46(5):733-40. DOI: 10.1016/0092-8674(86)90349-1. View

18.

Razzell W, Khorana H . Studies on polynucleotides. III. Enzymic degradation; substrate specificity and properties of snake venom phosphodiesterase. J Biol Chem. 1959; 234(8):2105-13. View

19.

Fielding P, LUNT M . A new deoxyribonuclease activity from bacteria infected with T5 bacteriophage. FEBS Lett. 1969; 5(3):214-217. DOI: 10.1016/0014-5793(69)80335-2. View

20.

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View