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 (> 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.

• Authors: Lawrence F Povirk, Tong Zhou, Ruizhe Zhou, Morton J Cowan, Steven M Yannone
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 282 Issue 6 Pg. 3547-58 (Feb 09 2007) ISSN: 0021-9258 [Print] United States
• PMID: 17121861 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• DNA-Binding Proteins
• Free Radicals
• Glycolates
• Nuclear Proteins
• Bleomycin
• DNA
• Zinostatin
• DNA-Activated Protein Kinase
• DCLRE1C protein, human
• Endodeoxyribonucleases
• Endonucleases
• phosphoglycolate

Topics

• Bleomycin (adverse effects)
• Cell Line
• DNA (drug effects, metabolism, radiation effects)
• DNA Damage
• DNA Repair (physiology)
• DNA-Activated Protein Kinase (metabolism)
• DNA-Binding Proteins
• Endodeoxyribonucleases (metabolism)
• Endonucleases
• Free Radicals (adverse effects)
• Glycolates (metabolism, radiation effects)
• Nuclear Proteins (genetics, physiology)
• RNA Processing, Post-Transcriptional (drug effects, radiation effects)
• X-Rays (adverse effects)
• Zinostatin (adverse effects)