Abstract
ARTEMIS is a member of the metallo-β-lactamase protein family. ARTEMIS has endonuclease activity at DNA hairpins and at 5'- and 3'-DNA overhangs of duplex DNA, and this endonucleolytic activity is dependent upon DNA-PKcs. There has been uncertainty about whether ARTEMIS also has 5'-exonuclease activity on single-stranded DNA and 5'-overhangs, because this 5'-exonuclease is not dependent upon DNA-PKcs. Here, we show that the 5'-exonuclease and the endonuclease activities co-purify. Second, we show that a point mutant of ARTEMIS at a putative active site residue (H115A) markedly reduces both the endonuclease activity and the 5'-exonuclease activity. Third, divalent cation effects on the 5'-exonuclease and the endonuclease parallel one another. Fourth, both the endonuclease activity and 5'-exonuclease activity of ARTEMIS can be blocked in parallel by small molecule inhibitors, which do not block unrelated nucleases. We conclude that the 5'-exonuclease is intrinsic to ARTEMIS, making it relevant to the role of ARTEMIS in nonhomologous DNA end joining.
Highlights
DNA-PKcs stimulates the endonucleolytic activities of ARTEMIS but not the 5Ј-exonuclease activity
ARTEMIS Endonuclease and 5Ј-Exonuclease Activities Copurify with WT ARTEMIS Protein—ARTEMIS has a well documented set of endonuclease activities, all of which are stimulated by DNA-PKcs (3)
The 5Ј-exonuclease activity is not stimulated by DNA-PKcs (3), and for this reason, we wondered whether the 5Ј-exonuclease is intrinsic to ARTEMIS
Summary
DNA-PKcs stimulates the endonucleolytic activities of ARTEMIS but not the 5Ј-exonuclease activity. All of the endonuclease activities of ARTEMIS on duplex DNA ends can be unified by a model invoking binding to the single- to double-stranded junction and occupying 4 nt along the single-stranded portion, followed by nicking preferentially 3Ј of those 4 nt (3). Similar to other members of the metallo--lactamase family, it is thought that ARTEMIS requires the binding of divalent cations to two co-catalytic portions of the active site for catalysis (13). We present a point mutant of ARTEMIS created using a baculovirus expression system, a system that more readily permits purification away from host cell 5Ј-exonucleases This mutation markedly reduces both the endonuclease as well as the 5Ј-exonuclease activities of ARTEMIS.
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