Unifying the DNA End Processing Roles of the Artemis Nuclease: Ku-Dependent Artemis Resection at Blunt DNA Ends [Enzymology]

August 14th, 2015 by Chang, H. H. Y., Watanabe, G., Lieber, M. R.

Artemis is a member of the metallo-β-lactamase protein family of nucleases. It is essential in vertebrates because during V(D)J recombination, the RAG complex generates hairpins when it creates the double-strand breaks at V, D, and J segments, and Artemis is required to open the hairpins so that they can be joined. Artemis is a diverse endo- and exonuclease, and a unified model for its wide range of nuclease properties has been challenging. Here we show that Artemis resects iteratively into blunt DNA ends with an efficiency that reflects the AT-richness of the DNA end. GC-rich ends are not cut by Artemis alone, due to a requirement for DNA end breathing (and confirmed using fixed pseudo-Y structures). All DNA ends are cut when both DNA-PKcs and Ku accompany Artemis, but not when Ku is omitted. This is the first biochemical data demonstrating a Ku-dependence of Artemis action on DNA ends of any configuration. The action of Artemis at blunt DNA ends is slower than at overhangs, consistent with a requirement for a slow DNA end breathing step preceding the cut. The AT-sequence dependence, the order of strand cutting, the length of the cuts, and the Ku-dependence of Artemis action at blunt ends can be reconciled with the other nucleolytic properties of both Artemis and Artemis:DNA-PKcs in a model incorporating DNA end breathing of blunt ends to form transient single- to double-strand boundaries that have structural similarities to hairpins and fixed 5′ and 3′ overhangs.