Purification of Rad1 protein from Saccharomyces cerevisiae and further characterization of the Rad1/Rad10 endonuclease complex.
The yeast recombination and repair proteins Rad1 and Rad10 associate with a 1:1 stoichiometry to form a stable complex with a relative molecular mass of 190 kDa. This complex, which has previously been shown to degrade single-stranded DNA endonucleolytically, also cleaves supercoiled duplex DNA molecules. In this reaction, supercoiled (form I) molecules are rapidly converted to nicked, relaxed (form II) molecules, presumably as a result of nicking at transient single-stranded regions in the supercoiled DNA. At high enzyme concentrations, there is a slow conversion of the form II molecules to linear (form III) molecules. The Rad1/Rad10 endonuclease does not preferentially cleave UV-irradiated DNA and has no detectable exonuclease activity. The nuclease activity of the Rad1/Rad10 complex is consistent with the predicted roles of the RAD1 and RAD10 genes of Saccharomyces cerevisiae in both the incision events of nucleotide excision repair and the removal of nonhomologous 3' single strands during intrachromosomal recombination between repeated sequences. In these pathways, the specificity and reactivity of the Rad1/Rad10 endonuclease will probably be modulated by further protein-protein interactions.
Tomkinson
A E
AE
Laboratory of Molecular Pathology, University of Texas Southwestern Medical Center, Dallas 75235.
Bardwell
A J
AJ
Tappe
N
N
Ramos
W
W
Friedberg
E C
EC
D003001:Cloning, Molecular; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins; D003851:Deoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005800:Genes, Fungal; D015151:Immunoblotting; D007700:Kinetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015719:Single-Strand Specific DNA and RNA Endonucleases
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D003851:Deoxyribonucleases; D004720:Endonucleases; C069266:RAD1 protein, S cerevisiae; C064162:RAD10 protein, S cerevisiae; D015719:Single-Strand Specific DNA and RNA Endonucleases; D045643:DNA Repair Enzymes