Processing of 3'-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease.
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 (> or =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.
Povirk
Lawrence F
LF
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Zhou
Tong
T
Zhou
Ruizhe
R
Cowan
Morton J
MJ
Yannone
Steven M
SM
D001761:Bleomycin / Q000009:adverse effects; D002460:Cell Line; D004247:DNA / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004720:Endonucleases; D005609:Free Radicals / Q000009:adverse effects; D006016:Glycolates / Q000378:metabolism* / Q000528:radiation effects; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects / Q000528:radiation effects; D014965:X-Rays / Q000009:adverse effects; D009353:Zinostatin / Q000009:adverse effects
D004268:DNA-Binding Proteins; D005609:Free Radicals; D006016:Glycolates; D009687:Nuclear Proteins; D001761:Bleomycin; D004247:DNA; D009353:Zinostatin; D051747:DNA-Activated Protein Kinase; C426344:DCLRE1C protein, human; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C013193:phosphoglycolate