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Saathoff, Jan-Hinnerk; Kaeshammer, Lisa; Lammens, Katja; Byrne, Robert Thomas; Hopfner, Karl-Peter (2018): The bacterial Mre11-Rad50 homolog SbcCD cleaves opposing strands of DNA by two chemically distinct nuclease reactions. In: Nucleic Acids Research, Vol. 46, No. 21: pp. 11303-11314
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Abstract

The Mre11-Rad50 complex is a DNA double-strand break sensor that cleaves blocked DNA ends and hairpins by an ATP-dependent endo/exonuclease activity for subsequent repair. For that, Mre11-Rad50 complexes, including the Escherichia coli homolog SbcCD, can endonucleolytically cleave one or both strands near a protein block and process free DNA ends via a 3'-5' exonuclease, but a unified basis for these distinct activities is lacking. Here we analyzed DNA binding, ATPase and nuclease reactions on different DNA substrates. SbcCD clips terminal bases of both strands of the DNA end in the presence of ATP gamma S. It introduces a DNA double-strand break around 20-25 bp from a blocked end after multiple rounds of ATP hydrolysis in a reaction that correlates with local DNA meltability. Interestingly, we find that nuclease reactions on opposing strands are chemically distinct, leaving a 5' phosphate on one strand, but a 3' phosphate on the other strand. Collectively, our results identify an unexpected chemical variability of the nuclease, indicating that the complex is oriented at a free DNA end and facing a block with opposite polarity. This suggests a unified model for ATP-dependent endo-and exonuclease reactions at internal DNA near a block and at free DNA ends.