Evidence that the DNA endonuclease ARTEMIS also has intrinsic 5'-exonuclease activity.
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.
From the Departments of Pathology, Biochemistry and Molecular Biology, Biological Sciences, and Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Norris Comprehensive Cancer Center, Los Angeles, California 90089-9176.
Sicong
S
Li
Howard H
HH
Chang
Doris
D
Niewolik
Michael P
MP
Hedrick
Anthony B
AB
Pinkerton
Christian A
CA
Hassig
Klaus
K
Schwarz
Michael R
MR
Lieber
D002845:Chromatography; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D059766:DNA End-Joining Repair; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004720:Endonucleases; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009708:Nucleotidases / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D017354:Point Mutation; D014162:Transfection
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D004247:DNA; C426344:DCLRE1C protein, human; D004720:Endonucleases; D005090:Exodeoxyribonucleases; D003850:Deoxyribonuclease I; D009708:Nucleotidases
DNA Repair; Nonhomologous DNA End Joining; Nucleic Acid; Nucleic Acid Enzymology; Nucleic Acid Structure; Protein DNA-Interaction