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.

• PMID: 15590332
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Comparative Study | Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Fricke WM, Bastin-Shanower SA, Brill SJ

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