TI - Discussion . AB - Previously , we showed that ATM and Artemis are required for a subset of DNA DSB repair that occurs with slow kinetics by NHEJ in G0/G1 phase . Here , we demonstrate that ATM and Artemis are also required for repair of a similar sized subset of DSBs , which are also rejoined with slow kinetics in G2 . However , in G2 this process represents HR . Furthermore , this represents the major contribution of HR to DSB repair after IR in G2 phase , with the majority of DSBs being rejoined by NHEJ in G0/G1 and G2 . These findings demonstrate that ATM and Artemis can contribute to DSB rejoining by either NHEJ or HR depending on cell -cycle phase . Previous studies have provided evidence that ATM is required for atleast a component of HR ( Morrison et al , 2000 ; Shrivastav et al , 2009 ) , but this has not previously been shown for Artemis . HR has its major role in dealing with replication-associated events , either by promoting repair of replication-blocking lesions or by repairing one-sided DSBs that arise at collapsed replication forks ( Trenz et al , 2006 ; Hanada et al , 2007 ; Roseaulin et al , 2008 ) . Since Artemis - or ATM-defective cells do not manifest the normal genomic instability and marked cross-link sensitivity of HR-defective mutants , there is no evidence that Artemis or ATM contribute to replication-associated HR events . Consistent with this , Artemis is dispensable for spontaneous SCEs . Thus , ATM and Artemis are not core HR components . However , HR also repairs two-ended DSBs in G2 and it is this situation where we uncovered a function for ATM and Artemis . We have applied three conceptually different techniques to demonstrate the requirement of ATM and Artemis for IR -induced HR in G2 . First , we used the enumeration of gammaH2AX foci in combination with epistasis analysis using the ATM inhibitor KU55933 and double-siRNA approaches . This analysis showed that loss of ATM or Artemis confers a repair defect , which is similar and epistatic to that of Brca2 , Rad51 and Rad54 in G2 . Conclusions based on gammaH2AX foci analysis were confirmed by PCC analysis . Second , we developed a BrdU labelling technique to detect repair synthesis during HR in G2 , and demonstrated that ATM and Artemis are required for G2 phase HR . Third , we developed and exploited a technique to quantify SCE events arising in G2-irradiated cells . Finally , the observation that ATM and Artemis are required for efficient formation of RPA , Rad51 and ssDNA foci provides further evidence for their role in promoting IR -induced HR in G2 . In contrast to these results , we failed to detect any requirement for Artemis in an I-SceI assay . However , this assay differs from our G2-specific assays in several ways : (i) I-SceI -induced DSBs need resection for repair by HR , but do not need end processing and ( ii ) the I-SceI assay reflects HR that arises at all cell -cycle stages including S-phase . Indeed , the fact that ATR , rather than ATM , has a larger role in this process may indicate that events in S-phase predominate where ATR , rather than ATM , may activate end resection at a DSB ( Jazayeri et al , 2006 ) . Our gammaH2AX , PCC , BrdU and RPA/Rad51 assays monitor the repair of IR -induced DSBs that arise in G2-phase , and our findings suggest that it is these HR events that require Artemis and ATM . Moreover , it is difficult to analyse specific subsets of DSBs by the I-SceI method . Thus , although the I-SceI approach can be informative , it has limitations and the novel pathway described here cannot be readily monitored by this assay . Previous studies have shown that the kinetics for IR -induced DSB repair in G1 exhibit a fast and a slow component ( Lobrich et al , 1995 ; Wu et al , 2008 ) . The fast component removes the majority of DSBs within the first 2 h and is strongly compromised in mutants of the NHEJ pathway ( Rothkamm et al , 2003 ; Kuhne et al , 2004 ) . The slow component in G1 represents a sub-pathway of NHEJ involving ATM and Artemis ( Riballo et al , 2004 ) . Previous studies by our group and others failed to observe a repair defect in HR-deficient mouse cells in G2-phase ( Kruger et al , 2004 ; Wu et al , 2008 ) . However , these studies used PFGE analysis , which necessitates high-radiation doses that lead to massive apoptosis in MEFs ( data not shown ) . Here , by using low-radiation doses compatible with cell survival , we show that DSB-repair kinetics in G2 are biphasic , as in G1 , and that the fast component in G2 represents NHEJ and accounts for the majority of DSB-repair events . However , the slow component of DSB repair in G2 represents HR involving Rad51 , Rad54 , Brca2 and , unexpectedly , ATM and Artemis . Thus , ATM and Artemis are involved in the slow component of DSB repair , which accounts for 15-20% of IR -induced DSBs in G1 and in G2 . However , these DSBs are subsequently repaired by distinct repair pathways . We recently showed that the DSBs requiring ATM for repair in G0/G1 are localized to heterochromatic DNA and that the requirement can be relieved by siRNA of KAP-1 , a heterochromatic component and ATM SUBstrate . We proposed that ATM -dependent PHOSphorylation of KAP-1 specifically promotes repair of DSBs located within the heterochromatin ( Goodarzi et al , 2008 ) . Here , we demonstrate that depleting KAP-1 relieves the ATM -dependent DSB-repair defect in G2 as in G1 . This finding is supported by an analysis of the localization of RPA foci and ATM -dependent DSBs . Since KAP-1 is randomly localized in G2-phase , the random localization of RPA foci and ATM -dependent DSBs does not itself provide evidence for heterochromatic localization . However , the striking change in localization between G1 - and G2-phase , which correlates with the different KAP-1 localization , is strongly supportive of the notion that HR occurs at heterochromatic DNA regions . Barlow et al recently reported that IR -induced DSBs are efficiently processed for HR in G1 in contrast to restriction endonuclease -induced DSBs . It has been suggested that this may result from the difference in modifying IR -induced versus endonuclease -induced DSBs prior to repair ( Kanaar et al , 2008 ; Wyman et al , 2008 ) . The differential response uncovered in our study reflects the processing of IR -induced heterochromatic versus euchromatic DSBs . Collectively , these findings suggest that only selective types of DSBs dependent upon their nature or localization may undergo resection , and further work is required to define the precise factors regulating whether or not resection occurs at a DSB . Our finding that Artemis is required for efficient resection during IR -induced HR might suggest that it represents the enzyme carrying out resection . However , the observation that both spontaneous SCE levels and I-SceI -induced HR do not require Artemis shows that resection can occur in the absence of Artemis . Moreover , we show that Rad51 foci formation after IR -induced HR in G2 requires CtIP , a factor , which promotes end resection during HR by interacting with the exonuclease Mre11 ( Sartori et al , 2007 ; Huertas et al , 2008 ) . Finally , the repair defect of Artemis-deficient cells cannot be complemented with an endonuclease -deficient Artemis construct , providing direct evidence that Artemis promotes HR as an endonuclease . An intriguing model is that Artemis is required to remove lesions or secondary structures , which arise in heterochromatic DNA regions that might otherwise inhibit resection . We have observed here that the role of Artemis in HR is independent of DNA-PK . During NHEJ , Artemis acquires endonuclease activity following DNA-PK auto-PHOSphorylation at DSB termini with hairpins or single-stranded DNA overhangs ( Goodarzi et al , 2006 ) . Notably , we showed that PHOSphorylation of Artemis by DNA-PK was dispensable for nuclease activity ( Goodarzi et al , 2006 ) , consistent with our observation here that DNA-PK is dispensable for Artemis endonuclease activity during HR . It is possible that the proposed requirement for DNA-PK to remodel the DNA end for Artemis during NHEJ ( Goodarzi et al , 2006 ) can be bypassed in some way by one or more factors during the process of HR . Based on our findings , we suggest the following model ( Figure 10 ) : The majority of IR -induced DSBs ( ~80% ) are repaired by NHEJ with fast kinetics in G1 and in G2 , independently of ATM and Artemis . However , a subset of DSBs in G1 and G2 is repaired more slowly and requires ATM and Artemis . This slowly repairing sub-fraction of breaks is channelled into NHEJ in G1 and into HR in G2 , thus requiring either the classical NHEJ or HR factors in addition to ATM and Artemis . Moreover , our finding that Artemis-deficient cells show impaired formation of ssDNA during IR -induced HR , together with the evidence that Artemis endonuclease is required for efficient DSB repair , suggests that Artemis promotes the processing step of DSB repair , which may be a prerequisite for resection of IR -induced DSBs . Finally , the observation that KAP-1 relieves the requirement for ATM for repair suggests that IR -induced HR in G2 repairs DSBs associated with heterochromatin .