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

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Previous work showed that, in the presence of 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' {yields} 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 ... continued below

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Povirk, Lawrence; Yannone, Steven M.; Khan, Imran S.; Zhou, Rui-Zhe; Zhou, Tong; Valerie, Kristoffer et al. February 18, 2008.

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Previous work showed that, in the presence of 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' {yields} 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 nucleotides 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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  • Journal Name: Nucleic Acids Research

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  • Report No.: LBNL-423E
  • Grant Number: DE-AC02-05CH11231
  • Office of Scientific & Technical Information Report Number: 934731
  • Archival Resource Key: ark:/67531/metadc896534

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  • February 18, 2008

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  • Sept. 27, 2016, 1:39 a.m.

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  • Sept. 30, 2016, 6:22 p.m.

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Povirk, Lawrence; Yannone, Steven M.; Khan, Imran S.; Zhou, Rui-Zhe; Zhou, Tong; Valerie, Kristoffer et al. Coordinateendonucleolytic 5' and 3' trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase, article, February 18, 2008; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc896534/: accessed November 15, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.