Nucleic Acids Research Advance Access originally published online on April 25, 2008
Nucleic Acids Research 2008 36(10):3354-3365; doi:10.1093/nar/gkn205
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Nucleic Acids Research, 2008, Vol. 36, No. 10 3354-3365
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Coordinate 5' and 3' endonucleolytic trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase
1Life Sciences Division, Department of Molecular Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, 2Department of Pharmacology and Toxicology and 3Department of Radiation Oncology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA
*To whom correspondence should be addressed. Tel: +1 804 828 9640; Fax: +1 804 828 8079; Email: lpovirk{at}vcu.edu
Received February 13, 2008. Revised April 7, 2008. Accepted April 7, 2008.
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.
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