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.

Nonhomologous end joining: A good solution for bad ends

“…6B). For example, Artemis and Metnase can excise the same 3′phosphoglycolate terminus as Tdp1/PNKP, except when nucleases are used more flanking DNA is lost and the products of excision are heterogeneous [78, 114, 135]. Since damage is removed as part of a patch (i.e.…”

Involvement of p53 in the Repair of DNA Double Strand Breaks: Multifaceted Roles of p53 in Homologous Recombination Repair (HRR) and Non-Homologous End Joining (NHEJ)

“…TDP1 removes glycolate residues from 3′ ends followed by additional processing by PNKP to remove the resulting 3′-phosphates (Zhou et al 2009). Recently, Metnase was shown to endonucleolytically trim 3′-overhangs greater than 3 bp on DNA duplexes, although its lack of activity when added to NHEJ-competent extracts casts some doubt on such a role in vivo (Mohapatra et al 2013). The various enzymes involved in the NHEJ pathway have a high flexibility in binding to DNA lesions, allowing them to interact or bind with a plethora of DNA end structures.…”

End-processing nucleases and phosphodiesterases: An elite supporting cast for the non-homologous end joining pathway of DNA double-strand break repair

“…Overall, Metnase exhibits trimming abilities similar to those of Artemis and can act to remove modified bases and blocked termini in some contexts. However, in an extract-based NHEJ model, addition of exogenous Artemis resulted in trimming and subsequent ligation of a DSB with a blocked 3´ overhang, but addition of Metnase did not, even though the same structure was a Metnase substrate as naked DNA [86]. This result casts doubt on a role for Metnase in resolving blocked DSB ends, as it suggests that presence of other NHEJ proteins may hinder rather than promote its endonuclease activity.…”

“…Mohapatra et al showed that Metnase trims overhangs that terminate in 3´-PGs, and in some cases the 3´-PG stimulated cleavage by Metnase and altered its specificity. However, the presence of a thymine glycol in a short 3´ overhang hindered Metnase's trimming ability in the immediate vicinity of the modified base although it could trim the same modified end in a 7-base overhang [86]. Overall, Metnase exhibits trimming abilities similar to those of Artemis and can act to remove modified bases and blocked termini in some contexts.…”

“…It has been found to play varied roles in chromosomal decatenation [82], DNA integration [83], restart of stalled replication forks [84] and in NHEJ repair [85,86]. A recent study showed that the Asn-610 residue within the catalytic domain of Metnase plays an important role in ssDNA binding which is essential for both single-strand overhang cleavage and also for the functions of Metnase in DSB repair and replication fork restart [87].…”