TI - KAP-1 -dependent heterochromatin correlates with G2-phase DSB repair by HR . AB - We recently showed that in G0/G1-phase ATM facilitates a component of NHEJ by regulating chromatin modifications in heterochromatic regions ( Goodarzi et al , 2008 ) . DSBs that remain unrepaired in ATM-defective or inhibited cells are enriched at densely staining DAPI chromocentres , which can be visualized in murine NIH3T3 cells . Knockdown of KAP-1 , a heterochromatic building factor that is PHOSphorylated by ATM , relieves the requirement for ATM for DSB repair ( Ziv et al , 2006 ; Goodarzi et al , 2008 ) . In our examination of G0/G1 cells , we carried out extensive analysis to verify published evidence that murine chromocentres represent heterochromatic DNA . Published , as well as our own , data suggest that this is not the case in G2 ( Fischle et al , 2005 ; Goodarzi et al , 2009 ) . Whereas the overall level of KAP-1 is similar in G1 and G2 , the amount of KAP-1 localized to the densely staining DAPI regions is strongly reduced in G2 compared with G1 ( Figure 9A and Supplementary Figure 8 ) . Thus , the organization of chromatin is distinct in G2 phase and condensed DAPI regions represent sites of chromosome condensation prior to mitosis rather than heterochromatic chromocentres ( Goodarzi et al , 2009 ) . The localization of the gammaH2AX foci , which remain in the absence of ATM at 8 h post-IR , correlates with the distribution of KAP-1 in both cell -cycle phases . Although this distribution parallels the intensity of DAPI staining in G2 phase ( ie reflects DNA distribution ) , the correlating change in the localization of KAP-1 - and ATM -dependent gammaH2AX foci from G1 to G2 is marked . Further , RPA foci present at 2 h post-IR in WT cells demonstrated a localization that also correlates with KAP-1 distribution ( Figure 9A and Supplementary Figure 8 ) . Importantly , we also examined the impact of knockdown of KAP-1 in G2 phase cells and found that it relieves the ATM -dependent DSB-repair defect in G2 as in G1 phase ( Figure 9B and C ) , that is , DSBs , which persist in an ATM -inhibited ( Figure 9B ) or ATM-downregulated cell line (Figure 9C) , can be repaired if KAP-1 is depleted . This strongly suggests that HR occurs at KAP-1 -dependent heterochromatin regions .