Coordinate 5′ and 3′ endonucleolytic trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase

Abstract

Previous work showed that, in the presence of DNA-dependent protein kinase (DNA-PK), Artemis slowly trims 3′-phosphoglycolate-terminated blunt ends. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. In the absence of DNA-PK, Artemis catalyzed extensive 5′→3′ exonucleolytic resection of double-stranded DNA. This resection required a 5′-phosphate, but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3′ overhang. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent and did not require a 5′-phosphate. For a blunt end with either a 3′-phosphoglycolate or 3′-hydroxyl terminus, endonucleolytic trimming of 2–4 nucleotides from the 3′-terminal strand was accompanied by trimming of 6 nt from the 5′-terminal strand. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5′-terminal strand, resulting in short 3′ overhangs that are trimmed endonucleolytically. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase-mediated patching and ligation, with minimal loss of terminal sequence. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.

Unifying the DNA End-processing Roles of the Artemis Nuclease

“…However, the action of Artemis at blunt DNA ends (and in a DNA-PKcs-independent manner) complicated this simple model (18). Here we confirmed the action at blunt ends, but we show that, like the other endonucleolytic actions of Artemis, this too is dependent on DNA-PKcs.…”

“…One study also described endonuclease action by Artemis alone in Mg 2ϩ solutions at blunt DNA ends (18). But this latter finding was curious because no other laboratory has found endonuclease activity of Artemis alone under Mg 2ϩ conditions.…”

Nonhomologous DNA end-joining for repair of DNA double-strand breaks

“…When ionizing radiation-induced DSBs bear a 3'-PG terminus (88)(89)(90), for example, these DNA ends are unable to undergo ligation since this step requires a 3'-hydroxyl on one end and a 5'-phosphate on the other. TDP1 is able to remove these 3' modifications; however, TDP1 mutant cells are only marginally radiosensitive compared to Artemis mutants, and it has been demonstrated biochemically that the Artemis·DNA-PKcs complex is able to process these ends (91,92). This suggests that Artemis can work with, or in place of TDP1 to repair the large number of DSBs that can occur following radiation exposure.…”

Limited Repair of Critical DNA Damage in Cells Exposed to Low Dose Radiation

“…This stage of processing in the repair by NHEJ is believed to be responsible for errors resulting from losses and additions of nucleotides at the sites of DNA DSB rejoining (Mahaney et al, 2009;Lieber, 2010). In the processing of broken DNA ends, the following enzymes are involved: the nuclease Artemis (Yannone et al, 2008), the DNA polymerases of the family X (Pol and Pol ) (Moon et al, 2007), and the terminal deoxynucleotidyl transferase (Mahaney et al, 2009), as well as polynucleotide kinase having both the kinase and phosphatase activities (Bernstein et al, 2008). Also, some other substances participate in the process, such as aprataxin (APTX) which catalyzes the removal of AMP groups at the 5 'ends of the DSB (Rass et al, 2008), the PALF-factor, and the protein cernunnos (XLF), which interact with XRCC4 in the activation of the DNA-ligase complex XRCC4/DNK-ligase IV/XLF Yano et al, 2009).…”