Chromosoma (1999) 108:44–51  Springer-Verlag 1999 Detection of poly(ADP-ribose) synthesis in Drosophila testes upon g-irradiation Susanne Lankenau, Alexander Bürkle 1 , Dirk-Henner Lankenau 2 1 Department of Tumor Virology, Deutsches Krebsforschungszentrum Heidelberg, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany 2 Department of Developmental Genetics, Deutsches Krebsforschungszentrum Heidelberg, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Received: 12 October 1998; in revised form: 21 December 1998 / Accepted: 23 December 1998 Edited by: P. Becker Correspondence to: D.-H. Lankenau e-mail: d.lankenau@dkfz-heidelberg.de Abstract. Poly(ADP-ribose) polymerase (PARP) activi- ty is widespread among eukaryotes. Upon DNA damage PARP binds to DNA strand breaks and transfers ADP-ri- bose residues from NAD + to acceptor proteins and to ADP-ribosyl protein adducts. This leads to branched polymers of protein-coupled poly(ADP-ribose) (pADPr). Because the germline of Drosophila has recently become important in the study of DNA double-strand break repair (DSBR) as opposed to somatic DSBR we tested whether the catalytic activity of PARP can be stimulated by g-ir- radiation during Drosophila spermatogenesis. Using anti- bodies against pADPr we detected a significant increase in PARP activity in male germline cells during spermato- genesis upon g-irradiation. Different stages of spermato- genesis revealed different subnuclear localization patterns of pADPr. In premeiotic and postmeiotic cells pADPr lo- calized in a pattern overlapping with lamin and topoisom- erase II at the nuclear rim. In primary spermatocytes pADPr is associated with three loci corresponding to the chromosomes at the nuclear periphery. Introduction Poly(ADP-ribosyl)ation of nuclear proteins in eukaryot- ic organisms is a reversible modification reaction that alters the structure and function of proteins (De Murcia and Menissier de Murcia 1994; Lindahl et al. 1995; Bür- kle 1998; Jeggo 1998). The reaction is catalyzed by at least two enzymes, poly(ADP-ribose) polymerase (PARP), and tankyrase (Smith et al. 1998). One of the first biochemical responses to DNA strand interruptions caused by ionizing radiation is the binding of PARP to the end of double-stranded DNA. This triggers the cata- lytic function of PARP, i.e., the successive transfer of ADP-ribose residues from NAD + to acceptor proteins and to ADP-ribosyl protein adducts (Althaus and Richter 1987; De Murcia and Menissier de Murcia 1994; Lindahl et al. 1995; Bürkle 1998, Malanga et al. 1998). PARP- bound polymers may act as a DNA decoy attracting se- lected proteins from the vicinity of DNA strand breaks through strong noncovalent interactions, and thereby pre- pare the damaged DNA for repair (Realini and Althaus 1992). Recently, a PARP gene was identified in Drosophila melanogaster (Uchida et al. 1993; Hanai et al. 1998) as well as the expressed sequence tag of a putative tankyrase (Smith et al. 1998). Because the germline of Drosophila has recently become important in the study of double- strand break repair (DSBR) (using mainly a homologous recombination mechanism) as opposed to somatic DSBR (where other repair mechanisms dominate) we tested whether the catalytic activity of PARP can be stimulated by g-irradiation-induced DNA strand breaks during Dro- sophila spermatogenesis (Gloor et al. 1991; Nassif et al. 1994; Lankenau 1995; Gloor and Lankenau 1998; Lankenau and Gloor 1998). We applied 83 Gy/10 min to dissected testes from Drosophila. Mammalian cells yield approximately 70 DSBs, and 600 to 1,000 single- strand breaks per gray per cell (Blöcher 1982; Ward 1990; Friedberg et al. 1995). Within seconds after inflic- tion of DNA damage, a single PARP molecule can as- sume a porcupine-like structure with up to 28 polymers of different sizes and structural complexities each con- taining branching points at an average of about 40 ADP-ribose units. Using monoclonal (Kawamitsu et al. 1984) as well as polyclonal (Affar et al. 1998) antibodies to poly(ADP-ribose) (pADPr) we detected increasing lev- els of poly(ADP-ribosyl)ation in spermatogonia, primary spermatocytes and postmeiotic stages of male gameto- genesis upon increasing doses of g-irradiation. At some stages of spermatogenesis we found an overlapping sub- nuclear distribution of pADPr with lamin and topoisome- rase II at the nuclear rim. The results encourage attempts to include PARP function in the ongoing analysis of dif- ferences between DSBR in the germline versus somatic DSBR mechanisms.