Substrate specificity of the Saccharomyces cerevisiae Mus81-Mms4 endonuclease.
Mus81-Mms4/Eme1 is a conserved structure-specific endonuclease that functions in mitotic and meiotic recombination. It has been difficult to identify a single preferred substrate of this nuclease because it is active on a variety of DNA structures. In addition, it has been suggested that the specificity of the recombinant protein may differ from that of the native enzyme. Here, we addressed these issues with respect to Mus81-Mms4 from S. cerevisiae. At low substrate concentrations, Mus81-Mms4 was active on any substrate containing a free end adjacent to the branchpoint. This includes 3'-flap (3'F), regressed leading strand replication fork (RLe), regressed lagging strand replication fork (RLa), and nicked Holliday junction (nHJ) substrates. Kinetic analysis was used to quantitate differences between substrates. High Kcat/Km values were obtained only for substrates with a 5'-end near the branchpoint (i.e., 3'F, RLe, and nHJ); 10-fold lower values were obtained for nicked duplex (nD) and RLa substrates. Substrates lacking any free ends at the branch point generated Kcat/Km values that were four orders of magnitude lower than those of the preferred substrates. Native Mus81-Mms4 was partially purified from yeast cells and found to retain its preference for 3'F over intact HJ substrates. Taken together, these results narrow the range of optimal substrates for Mus81-Mms4 and indicate that, at least for S. cerevisae, the native and recombinant enzymes display similar substrate specificities.
Fricke
William M
WM
Department of Molecular Biology and Biochemistry, Rutgers University, 679 Hoes Lane, Piscataway, NJ 08854, USA.
Bastin-Shanower
Suzanne A
SA
Brill
Steven J
SJ
D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D045585:Flap Endonucleases; D007700:Kinetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D015534:Trans-Activators / Q000378:metabolism*
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D004720:Endonucleases; D045585:Flap Endonucleases; C411624:MUS81 protein, S cerevisiae; C112337:MMS4 protein, S cerevisiae