Purification and characterization of exonuclease-free Artemis

Implications for DNA-PK-dependent processing of DNA termini in NHEJ-catalyzed DSB repair

Katherine S. Pawelczak, John Turchi

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Artemis is a member of the β-CASP family of nucleases in the metallo-β-lactamase superfamily of hydrolases. Artemis has been demonstrated to be involved in V(D)J-recombination and in the NHEJ-catalyzed repair of DNA DSBs. In vitro, both DNA-PK independent 5′-3′ exonuclease activities and DNA-PK dependent endonuclease activity have been attributed to Artemis, though mutational analysis of the Artemis active site only disrupts endonuclease activity. This suggests that either the enzyme contains two different active sites, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. Recombinant [His]6-Artemis was expressed in a Baculovirus insect-cell expression system and isolated using a three-column purification methodology. Exonuclease and endonuclease activities, the ability to be phosphorylated by DNA-PK, and Artemis antibody reactivity was monitored throughout the purification and to characterize final pools of protein preparation. Results demonstrated the co-elution of exonuclease and endonuclease activities on a Ni-agarose affinity column but separation of the two enzymatic activities upon fractionation on a hydroxyapatite column. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease.

Original languageEnglish
Pages (from-to)670-677
Number of pages8
JournalDNA Repair
Volume9
Issue number6
DOIs
StatePublished - Jun 4 2010

Fingerprint

Exonucleases
Endonucleases
Purification
Repair
DNA
Processing
His-His-His-His-His-His
Distillation columns
Catalytic Domain
DNA End-Joining Repair
V(D)J Recombination
Aptitude
Antibodies
Baculoviridae
Hydrolases
Durapatite
Fractionation
1,2-di-(4-sulfamidophenyl)-4-butylpyrazolidine-3,5-dione
Sepharose
Insects

Keywords

  • Artemis
  • DNA end processing
  • DNA-PK
  • NHEJ

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology
  • Medicine(all)

Cite this

Purification and characterization of exonuclease-free Artemis : Implications for DNA-PK-dependent processing of DNA termini in NHEJ-catalyzed DSB repair. / Pawelczak, Katherine S.; Turchi, John.

In: DNA Repair, Vol. 9, No. 6, 04.06.2010, p. 670-677.

Research output: Contribution to journalArticle

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