Human Topors, which was originally identified as cellular binding partner of DNA topoisomerase I and of p53, has recently been shown to function as an ubiquitin E3 ligase for p53 in a manner dependent on its N 0 -terminally located RING finger. Here, we demonstrate that Topors also enhances the conjugation of the small ubiquitin-like modifier 1 (SUMO-1) to p53 in vivo and in a reconstituted in vitro system. The Topors SUMO-1 E3 ligase activity does not depend upon its RING finger motif. In HeLa cells, Topors induced p53 sumoylation was accompanied by an increase in endogenous p53 protein levels. Furthermore, Topors enhances the sumoylation of a variety of other, yet unidentified, cellular proteins.

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Adeno-associated virus type 2 (AAV-2) is a human parvovirus that, for efficient reproduction, requires coinfection of its host cell with a helper virus such as adenovirus or herpesvirus (Berns & Linden, 1995). The 4n7 kb single-stranded DNA genome (Srivastava et al., 1983) consists of two open reading frames (ORFs) flanked by two 145 base pair inverted terminal repeats (ITRs). The ORF in the right half of the AAV genome encodes the three structural proteins, VP1, VP2 and VP3, which are transcribed from the p40 promoter. The ORF in the left half of the genome encodes four overlapping nonstructural proteins, termed Rep (Mendelson et al., 1986). The p5 promoter directs the synthesis of Rep78 and Rep68, while the p19 promoter drives the expression of Rep52 and Rep40. Rep78 and Rep68 are essential for AAV DNA replication (Tratschin et al., 1984) and regulation of AAV gene expression (Labow et al., 1986 ;Tratschin et al., 1986). They possess ATP-

Human Topors has originally been identified as binding partner of p53 and DNA topoisomerase I (TOP1). It can function as both an ubiquitin and SUMO-1 E3 ligase for p53. Here we demonstrate that Topors enhances the formation of high-molecular weight SUMO-1 conjugates of TOP1 in a reconstituted in vitro system and also in human osteosarcoma cells, similar to treatment with CPT. In contrast to the situation observed with p53, overall sumoylation levels were rather unaffected. Experiments with TOP1 point mutants strongly suggest that the high-molecular weight conjugates represent SUMO-1 chains formed on a limited number of SUMO-1 acceptor sites.