DNA and Chromosomes
Evidence That the DNA Endonuclease ARTEMIS also Has Intrinsic 5′-Exonuclease Activity*

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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.

DNA Repair
Nucleic Acid
Nucleic Acid Enzymology
Nucleic Acid Structure
Protein DNA-Interaction
Nonhomologous DNA End Joining

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*

This work was supported by a National Institutes of Health Grants R01 CA100504 and R01 CA51105 (to M. R. L.) and the BMBF (German Federal Ministry of Education & Research) Grant 01GM1111F (to K. S.).

2

The abbreviations used are:

    β-CASP

    β-lactamase, CPSF (cleavage and polyadenylation specificity factor), Artemis, Snm1, Pso2 domain

    NHEJ

    nonhomologous DNA end joining

    DNA-PKcs

    DNA-dependent protein kinase, catalytic subunit

    ssDNA

    single-stranded DNA

    Ni-NTA

    nickel-nitrilotriacetic acid.