Recognition of (dG)n.(dC)n sequences by endonuclease G. Characterization of the calf thymus nuclease.
We report the purification of endonuclease G (Ruiz-Carrillo, A., and Renaud, J. (1987) EMBO J. 6, 401-407) from calf thymus nuclei and whole tissue. The enzyme has been enriched 29,000-fold, and the activity was unambiguously identified with a 26-kDa protein after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native nuclease behaves as a 50-kDa species by gel filtration, suggesting that it is composed of two subunits, presumably identical. In terms of absolute amounts, endonuclease G (endo G) is a nuclear enzyme although it was also detected in purified mitochondria. Endo G is highly specific for (dG)n.(dC)n tracts in DNA, nicking either strand of relaxed substrates with similar kinetics. The sensitivity of the homopolymer tracts is proportional to their length (from n = 8 to 29), insofar as the flanking sequences are constant. However, the overall rate of cleavage is influenced by the composition of the flanking DNA. Minor cleavage sites contain shorter (dG)n.(dC)n clusters (n = 3-7). Endo G efficiently cleaves (dC)n but not (dG)n runs in single-stranded DNA, suggesting that it may recognize an asymmetric strand conformation of the homopolymer tracts. Endo G does not recognize other homo(co)-polymer sequences or cruciform structures in DNA.
Côté
J
J
Cancer Research Center, Laval University School of Medicine, Québec, Canada.
Renaud
J
J
Ruiz-Carrillo
A
A
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology; D002850:Chromatography, Gel; D004277:DNA, Single-Stranded / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D011489:Protein Denaturation; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*
D004277:DNA, Single-Stranded; D011089:Polydeoxyribonucleotides; C019530:poly(dC-dG); D004706:Endodeoxyribonucleases; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII; C052391:endonuclease G