IDENTIFICATION OF A 116 KDA PROTEIN ABLE TO BIND 1,3-BIS(2-CHLOROETHYL)-1-NITROSOUREA-DAMAGED DNA AS POLY(ADP-RIBOSE) POLYMERASE

SKI-I is a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant gliom a cell line and SK-MG-I is a BCNU-sensitive glioma cell line. Both cel l lines do not express O-6-methylguanine-DNA methyl transferase (MGMT) and exhibit comparable levels of 3-methyladenine DNA glycosylase. In order to detect DNA binding proteins involved in alternative DNA repai r mechanisms of BCNU damage, we performed Southwestern analysis using a DNA probe damaged with BCNU and nuclear protein extracts from SKI-1 and SK-MG-1 cell lines. Both cell lines express a protein of M(r) 116 000 that is able to bind to BCNU-damaged DNA with higher specificity t han to undamaged DNA. This protein was identified as poly(ADP-ribose) polymerase (PARP). Using glioma extracts depleted of PARP or using ant ibody to block the DNA binding domain of PARP no other protein binding to BCNU-treated probe was observed. Addition of methoxyamine, an inhi bitor of DNA strand breaks, led to a significant reduction of PARP bin ding to BCNU-treated DNA. BCNU treatment of both glioma cell lines led to reduced nicotinamide adenine dinucleotide levels, indicating activ ation of PARP. Thus, the recognition and binding of PARP to BCNU-induc ed DNA nicks with concomitant PARP activation may be important process es that are involved in the initial stage of DNA repair of BCNU lesion s in glial cells.