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

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 53 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 24 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.