Processing of 3'-Phosphoglycolate-Terminated DNA Double-StrandBreaks 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 functions on oxidatively modified double-strand break termini, its activity toward model DNA substrates, bearing either 3{prime}-hydroxyl or 3{prime}-phosphoglycolate moieties, was examined. A 3{prime}-phosphoglycolate had little effect on Artemis-mediated trimming of long 3{prime} overhangs (>9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3{prime}-phosphoglycolates on overhangs of 4-5 bases promoted selective Artemis-mediated trimming of a single 3{prime}-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{prime} overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was dependent upon Ku, DNA-dependent protein kinase, and ATP. Together, these data suggest that Artemis-mediated cleavage of 3{prime} overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3{prime} to the cleavage site. Shorter 3{prime}-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis, but much less efficiently. Consistent with the in vitro substrate specificity of Artemis, human cells lacking Artemis exhibited hypersensitivity to X-rays, bleomycin and neocarzinostatin, which all induce 3{prime}-phosphoglycolate-terminated double-strand breaks. Collectively, these results suggest that 3{prime}-phosphoglycolate termini and/or specific classes of DNA ends that arise from such blocked termini are relevant Artemis substrates in vivo.
- Research Organization:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Sponsoring Organization:
- USDOE Director, Office of Science. Office of Biological andEnvironmental Research. Life Sciences Division
- DOE Contract Number:
- DE-AC02-05CH11231
- OSTI ID:
- 923183
- Report Number(s):
- LBNL-59160; JBCHA3; R&D Project: L0423; BnR: 600303000; TRN: US200804%%1019
- Journal Information:
- Journal of Biological Chemistry, Vol. 282, Issue 6; Related Information: Journal Publication Date: 02/09/2007; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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