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 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.
Yannone
Steven M
SM
Life Sciences Division, Department of Molecular Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Khan
Imran S
IS
Zhou
Rui-Zhe
RZ
Zhou
Tong
T
Valerie
Kristoffer
K
Povirk
Lawrence F
LF
D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007106:Immune Sera / Q000494:pharmacology; D009154:Mutation; D010710:Phosphates / Q000737:chemistry
D007106:Immune Sera; D010710:Phosphates; D004247:DNA; D051747:DNA-Activated Protein Kinase; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases