TI - ATM and Artemis are required for IR -induced SCEs in G2 . AB - We sought further evidence for a role of ATM and Artemis in promoting G2 HR and studied the formation of SCEs as a well-established marker for HR events ( Sonoda et al , 1999 and Figure 4 ) . We grew HeLa cells in BrdU-containing medium for two cell cycles , added aphidicolin immediately before irradiation with 2 Gy to exclude the S-phase cells from analysis and collected metaphases up to 12 h post-IR . Pilot experiments had shown that G2 cells irradiated with 2 Gy enter mitosis between 8 and 12 h after irradiation ( data not shown and Deckbar et al , 2007 ) . Hence , this procedure monitors SCE formation arising from HR in G2-phase . We observed ~7 SCEs per cell in un-irradiated cells , which increased to ~14 SCEs per cell after IR treatment ( Figure 4 ) . Downregulation of Rad51 or Brca2 by siRNA led to significant reduction in the spontaneous SCE level and completely abolished the formation of radiation-induced SCEs ( Figure 4 ) . Spontaneous SCEs are likely produced during the preceding cell cycles when siRNA downregulation is ongoing but not yet optimal . Thus , the reduction observed after Rad51 or Brca2 depletion probably represents an underestimation . In contrast to the effects of Rad51 and Brca2 , neither spontaneous nor IR -induced SCE level is significantly affected by depleting Ku80 or Lig4 . Most importantly , downregulation of Artemis or ATM does not substantially affect the spontaneous SCE level , but nearly completely abolishes the increase observed after irradiation ( Figure 4 ) . Hence , Artemis and ATM play a major role in promoting HR of IR -induced DSBs in G2 , but are unlikely to represent core components of the HR machinery . We also examined whether Artemis and ATM are involved in HR using a plasmid-based I-SceI system ( Supplementary Figure 4 ) . We measured the frequency of reconstitution of a GFP reporter gene within a chromosomally integrated plasmid SUBstrate in transformed human fibroblasts with or without silencing of Artemis , ATR or Brca2 ( Supplementary Figure 4A ) , as previously described ( Pierce et al , 1999 ) . Transient expression of the I-SceI endonuclease generates a DSB , which can be repaired by gene conversion , yielding a functional GFP gene whose expression can be assessed by flow cytometry ( Supplementary Figure 4B ) . Consistent with previous studies ( Moynahan et al , 2001 ; Wang et al , 2004 ) , we observed significant reduction in I-SceI -induced HR after depletion of Brca2 or ATR , but failed to observe any effect in cells depleted for Artemis ( Supplementary Figure 4C ) . Moreover , there was only a small impact ( approximately one-third ) caused by inhibiting ATM ( Supplementary Figure 4C ) . The ATR dependency perhaps suggests that this assay preferentially monitors HR events occurring during S-phase . In any case , the lack of a pronounced dependency on ATM or Artemis confirms that these factors are not core HR enzymatic components .