title: Trimming of damaged 3' overhangs of DNA double-strand breaks by the Metnase and Artemis endonucleases.
abstract: Both Metnase and Artemis possess endonuclease activities that trim 3' overhangs of duplex DNA. To assess the potential of these enzymes for facilitating resolution of damaged ends during double-strand break rejoining, substrates bearing a variety of normal and structurally modified 3' overhangs were constructed, and treated either with Metnase or with Artemis plus DNA-dependent protein kinase (DNA-PK). Unlike Artemis, which trims long overhangs to 4-5 bases, cleavage by Metnase was more evenly distributed over the length of the overhang, but with significant sequence dependence. In many substrates, Metnase also induced marked cleavage in the double-stranded region within a few bases of the overhang. Like Artemis, Metnase efficiently trimmed overhangs terminated in 3'-phosphoglycolates (PGs), and in some cases the presence of 3'-PG stimulated cleavage and altered its specificity. The nonplanar base thymine glycol in a 3' overhang severely inhibited cleavage by Metnase in the vicinity of the modified base, while Artemis was less affected. Nevertheless, thymine glycol moieties could be removed by Metnase- or Artemis-mediated cleavage at sites farther from the terminus than the lesion itself. In in vitro end-joining systems based on human cell extracts, addition of Artemis, but not Metnase, effected robust trimming of an unligatable 3'-PG overhang, resulting in a dramatic stimulation of ligase IV- and XLF-dependent end joining. Thus, while both Metnase and Artemis are biochemically capable of resolving a variety of damaged DNA ends for the repair of complex double-strand breaks, Artemis appears to act more efficiently in the context of other nonhomologous end joining proteins.
authors.affiliation: Department of Pharmacology and Toxicology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Susovan
authors.initials: S
authors.lastname: Mohapatra
authors.forename: Steven M
authors.initials: SM
authors.lastname: Yannone
authors.forename: Suk-Hee
authors.initials: SH
authors.lastname: Lee
authors.forename: Robert A
authors.initials: RA
authors.lastname: Hromas
authors.forename: Konstantin
authors.initials: K
authors.lastname: Akopiants
authors.forename: Vijay
authors.initials: V
authors.lastname: Menon
authors.forename: Dale A
authors.initials: DA
authors.lastname: Ramsden
authors.forename: Lawrence F
authors.initials: LF
authors.lastname: Povirk
mesh_terms: D018736:DNA Adducts / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D011088:DNA Ligases / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004720:Endonucleases; D006016:Glycolates / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D059767:Recombinational DNA Repair; D013941:Thymine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D018736:DNA Adducts; D004268:DNA-Binding Proteins; D006016:Glycolates; C508551:NHEJ1 protein, human; D009687:Nuclear Proteins; C029389:thymine glycol; D011495:Histone-Lysine N-Methyltransferase; C510450:SETMAR protein, human; D051747:DNA-Activated Protein Kinase; C426344:DCLRE1C protein, human; D004720:Endonucleases; D011088:DNA Ligases; D045643:DNA Repair Enzymes; C013193:phosphoglycolate; D013941:Thymine