• 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • Our data supports the conclusion that the exonuclease activi


    Our data supports the conclusion that the exonuclease activity thought to be intrinsic to Artemis is not a component of the Artemis polypeptide. Many possibilities exist to explain the presence of the exonuclease activity in less-pure fractions of Artemis. It is possible that the exonuclease is simply a contaminating enzyme that is endogenously expressed in the insect Cy5 TSA and co-purifies with Artemis independent of any biologically relevant interaction. This possibility is supported by data presented where single-strand 5′–3′ exonuclease activity co-purifies with another DNA repair protein, [His]6-XPA, overexpressed in insect cells. Not only do these results support the hypothesis that the exonuclease is from SF9 cells, but it also raises the possibility that this exonuclease has an affinity for nickel–agarose resin, as both the XPA and Artemis were fractionated over this column first. This is not an unlikely scenario, as insect cells contain many endogenous nucleases and the 5′–3′ exonuclease SNM1 was also identified in D. melanogaster[32]. It is also possible that the exonuclease is endogenously associated with Artemis, and is a biologically relevant interaction. This intriguing possibility has yet to be investigated. The results presented here demonstrate that the exonuclease activity previously thought to be intrinsic to Artemis is separable from the DNA-PK and ATP dependent endonuclease activity of Artemis prompting further studies to clarify a role for exonuclease processing in NHEJ and the protein responsible for that activity.
    Conflict of interest
    Introduction Double-strand breaks (DSBs) can lead to genomic instability, which causes cancer and promotes aging. Cells maintain genomic stability by activating the DNA damage response after DSBs. As the first step of the cellular response to DSBs, the histone H2A variant H2AX located around DSB sites is phosphorylated on Ser139 (known as γH2AX) by the phosphatidylinositol-3 (PI3) kinase family proteins. Subsequently, γH2AX plays a critical role in DNA damage signaling, which induces cell-cycle checkpoints, apoptosis, and DSB repair [1], [2]. The PI3 kinase family includes DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), and ATM and Rad3-related (ATR). H2AX is phosphorylated by ATM and DNA-PK after DSBs, and by ATR after replication stress and SSB or UV [2]. Nonhomologous end joining (NHEJ) and homologous recombination (HR) are two major repair pathways of DSBs. Each pathway requires a set of repair factors [3]. Ku70 and Ku80 recognize and bind the DSB sites after DNA damage. Subsequently, the Ku70/80–DNA complex stabilizes and stimulates DNA-PK catalytic subunit (DNA-PKcs) to form the DNA-PK complex on the DSB sites. DNA-PK complex phosphorylates various proteins and promotes NHEJ [4]. Therefore, the absence of DNA-PK leads to increased HR efficiency after DSBs, which is consistent with the passive shunting of DSBs from NHEJ to HR [5]. However, several studies reported that DNA-PK inhibition causes a decrease in HR efficiency [6], [7]. In addition, recent studies have been reported that NHEJ consists of several separate pathways containing classical and alternative NHEJ [3], [8]. The classical NHEJ is well-known as Ku heterodimer and DNA-PKcs dependent pathway. The alternative NHEJ has been detectable in the absence of DNA-PKcs, Ku70 or Ku80, therefore this pathway is called DNA-PK independent or backup NHEJ [9]. These suggest the complicated regulation of DSB repair by DNA-PK but the molecular mechanism of which is still unknown. In addition, many studies reported the regulation mechanism of DSB repair pathways in mammalian cells, especially humans and mice. Therefore, the common DSB mechanism among vertebrates is still unknown. Medaka (Oryzias latipes) is used as a useful model in radiation biology [10], [11]. RIC1 strain was isolated as a radiation-sensitive mutant strain of Medaka via N-ethyl-N-nitrosourea ENU-mutagenesis screening [12]. Previously, we established four RIC1 cell lines from each single embryo and reported that RIC1 embryonic cells have a defect in γH2AX foci formation after γ-irradiation [13]. The result suggested that the ric1 gene is involved in the regulation of ATM or DNA-PK. In this study, we performed γH2AX focus assay and NHEJ and HR reporter assays after ATM or DNA-PK inhibitor treatment, and examined phosphorylated DNA-PKcs (Thr2609) and tumor suppressor p53 binding protein 1 (53BP1) focus after γ-irradiation. The results suggest a model that 53BP1 dependent NHEJ is an alternative mechanism to repair DSBs under the condition that decreases DNA-PK activity, which causes reduced HR efficiency.