Trimming of damaged 3' overhangs of DNA double-strand breaks by the Metnase and Artemis endonucleases

Susovan Mohapatra, Steven M. Yannone, Suk Hee Lee, Robert A. Hromas, Konstantin Akopiants, Vijay Menon, Dale A. Ramsden, Lawrence F. Povirk

Research output: Contribution to journalArticle

14 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)422-432
Number of pages11
JournalDNA Repair
Volume12
Issue number6
DOIs
StatePublished - Jun 1 2013

Keywords

  • Clustered DNA damage
  • Free radicals
  • Nonhomologous end joining
  • Oxidative damage

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Mohapatra, S., Yannone, S. M., Lee, S. H., Hromas, R. A., Akopiants, K., Menon, V., Ramsden, D. A., & Povirk, L. F. (2013). Trimming of damaged 3' overhangs of DNA double-strand breaks by the Metnase and Artemis endonucleases. DNA Repair, 12(6), 422-432. https://doi.org/10.1016/j.dnarep.2013.03.005