Open AccessPartial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions.
Author(s) -
Lorraine S. Symington,
Richard D. Kolodner
Publication year - 1985
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.82.21.7247
Subject(s) - holliday junction , cruciform , endonuclease , dna , nuclease , cleavage (geology) , saccharomyces cerevisiae , biology , restriction enzyme , biochemistry , bacteriophage , plasmid , gel electrophoresis , enzyme , microbiology and biotechnology , escherichia coli , homologous recombination , yeast , gene , history , paleontology , archaeology , fracture (geology)
An enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions was partially purified approximately 500- to 1000-fold by DEAE-cellulose chromatography, gel filtration on Sephacryl S300, and chromatography on single-stranded DNA-cellulose. The partially purified enzyme did not have any detectable nuclease activity when tested with single-stranded or double-stranded bacteriophage T7 substrate DNA and did not have detectable endonuclease activity when tested with bacteriophage M13 viral DNA or plasmid pBR322 covalently closed circular DNA. Analysis of the products of the cruciform cleavage reaction by electrophoresis on polyacrylamide gels under denaturing conditions revealed that the cruciform structure was cleaved at either of two sites present in the stem of the cruciform and was not cleaved at the end of the stem. The cruciform cleavage enzyme was able to cleave the Holliday junction present in bacteriophage G4 figure-8 molecules. Eighty percent of these Holliday junctions were cleaved in the proper orientation to generate intact chromosomes during genetic recombination.