Processing of 3′-phosphoglycolate-terminated DNA double strand breaks by artemis nuclease

Abstract

The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double strand breaks. To assess the possibility that Artemis acts on oxidatively modified double strand break termini, its activity toward model DNA substrates, bearing either 3′-hydroxyl or 3′-phosphoglycolate moieties, was examined. A 3′-phosphoglycolate had little effect on Artemis-mediated trimming of long 3′ overhangs (>9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3′-phosphoglycolates on overhangs of 4-5 bases promoted Artemis-mediated removal of a single 3′-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3′ overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was completely dependent on DNA-dependent protein kinase and ATP and was largely dependent on Ku, which markedly stimulated Artemis activity toward all 3′ overhangs. Together, these data suggest that efficient Artemis-mediated cleavage of 3′ overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3′ to the cleavage site, as well as 2 unpaired nucleotides 5′ to the cleavage site. Shorter 3′-phosphoglycolate- terminated overhangs and blunt ends were also processed by Artemis but much more slowly. Consistent with a role for Artemis in repair of terminally blocked double strand breaks in vivo, human cells lacking Artemis exhibited hypersensitivity to x-rays, bleomycin, and neocarzinostatin, which all induce 3′-phosphoglycolate-terminated double strand breaks.