TI - Results PARP-1 interacts with ATM in vivo and ATM is modified by poly ( ADP-ribosyl ) ation . AB - Previous studies have shown that PARP-1 and ATM double deficient mice are embryonic lethal very early during development , suggesting that the two proteins together are needed for the every day life of the animal [12] . In the present study our principal aim was to test whether these two proteins interact ( both physical and functionally ) in the response to gamma-radiation . Figure 1A shows an ATM co-immunoprecipitation study using G361 cells ( HT44 , an ATM deficient cell line , was used as a negative control ) . ATM complexes were immunoprecipitated from nuclear extracts using an antibody against ATM (SYR 10G3/1) and the presence of PARP-1 was tested by immunoblot analysis using an anti-PARP-1 antibody . ATM form a tight complex with PARP-1 ( 1A , upper panels ) . Reciprocal immunoprecipitation experiments confirmed the previous observation ( not shown ) . Interestingly , this complex was much more evident after DNA damage infringed with either the single alkylating agent N-methyl-N-nitrosourea ( MNU , 2 mM ) or 10 Gy of gamma-irradiation . The interaction was direct and not mediated by DNA since the presence of ethidium bromide was not effective in abolishing the formation of the complex and the specificity of the pull-down was confirmed by the lack of co-immunoprecipitation using an IgG control ( not shown ) . These results were confirmed by co-localisation studies with confocal microscopy , where after gamma-irradiation the number of co-localised ATM/PARP-1 foci ( yellow ) increased respect to untreated cells . Both ATM and PARP-1 are localised in foci after DNA damage . In order to check whether ATM was modified or not by PARP-1 following DNA damage , the modification of ATM by PARP-1 was analysed in a time course experiment using the same antibody to immunoprecipitate ATM ( figure 2A ) . ATM is , indeed , modified by poly ( ADP-rybosil ) ation and this modification increases during DNA damage , reaching a maximum after 30 min and then start to decrease . Polymer signal correspond to ATM molecular weight . Again , confocal microscopy confirmed the co-localisation of ATM and poly ( ADP ) ribose after ionizing radiation ( figure 2B ) . In conclusion , these results are the first indication that ATM is physically associated to PARP-1 and is a SUBstrate for this enzyme , co-localizing in the same foci after DNA damage .