Dissociation of the recombination control and the sequence-specific transactivation function of P53

Abstract

Recently, we described a new biological function of p53 in inhibiting recombination processes when encountering mismatches in heteroduplexes (DudenhoÈ er et al., 1998). Here, we characterized protein domains of p53 participating in this process by in vitro analysis of mutated p53 proteins, and by applying our SV40-based assay system on monkey cells, which express di erent p53 variants. We present evidence that both binding of arti®cial recombination intermediates and p53-dependent recombination control require an intact p53 core and the oligomerization domain, strongly suggesting that the recognition of DNA undergoing recombination represents an essential step of this genomic surveillance mechanism. Further analyses indicated a role of the C-terminus in negatively regulating recombination control, an e ect which can be neutralized by concurrent mismatch recognition. p53 lacking the oligomerization domain totally lost its ability to suppress homologous recombination. The cancer-related mutant p53(273H) was also signi®cantly defective in this function, although we observed only twofold reductions in the corresponding transactivation activities on p53-response elements in episomal constructs. HDM2, an inhibitor of p53's transcriptional and growth regulatory activities, interfered with the inhibition of DNA exchange processes by p53 only weakly. Thus, functions of p53 in recombination control can be structurally dissociated from p53-dependent transcriptional transactivation.

Role of heteroduplex joints in the functional interactions between human Rad51 and wild-type p53

“…Recently, we established a functional link between the recognition of heterologies on recombination intermediates and the inhibition of homologous recombination processes by wild-type p53 (DudenhoÈ er et al, 1998(DudenhoÈ er et al, , 1999. Given that Rad51 represents the initial strand transferase in eukaryotes (Benson et al, 1994;Sung and Robberson, 1995;Gupta et al, 1997) we studied strand exchange by human Rad51 (hRad51) in the presence or absence of human p53.…”

“…The activity of the mismatch repair system has been shown to inhibit recombination between diverged sequences (reviewed in Modrich and Lahue, 1996). In our previous work, we suggested that p53 is a key factor of a similar control mechanism to ensure the ®delity of recombination (DudenhoÈ er et al, 1998). According to these data, p53 inhibits homologous recombination in primate cells, especially when A-G and C-T mismatches are generated on heteroduplexes after strand exchange.…”

“…Several studies pointed towards a role of p53 in nucleotide excision repair possibly via interactions with the helicase subunits of the dual transcription/nucleotide excision repair factor TFIIH (Smith et al, 1995;Wang et al, 1995;Leveillard et al, 1996;Mirzayans et al, 1996;Ford and Hanawalt, 1997) or indirectly by suppressing a recombinational repair bypass mechanism (Ishizaki et al, 1994;Cleaver et al, 1999). During the last years, it was reported from di erent laboratories that p53 inhibits spontaneous homologous recombination events in mitotically growing cells (WiesmuÈ ller et al, 1996;Bertrand et al, 1997;Mekeel et al, 1997;DudenhoÈ er et al, 1998). Importantly, this genome stabilizing function is unrelated to transcriptional or cell-cycle regulatory activities of p53, and must be considered an independent activity, which contributes to tumor suppression (Saintigny et al, 1999;DudenhoÈ er et al, 1999).…”

“…During the last years, it was reported from di erent laboratories that p53 inhibits spontaneous homologous recombination events in mitotically growing cells (WiesmuÈ ller et al, 1996;Bertrand et al, 1997;Mekeel et al, 1997;DudenhoÈ er et al, 1998). Importantly, this genome stabilizing function is unrelated to transcriptional or cell-cycle regulatory activities of p53, and must be considered an independent activity, which contributes to tumor suppression (Saintigny et al, 1999;DudenhoÈ er et al, 1999).…”

“…Additionally, p53 shows high binding a nities in vitro for 3-stranded recombination intermediates, especially when encompassing these mispairings. Concerning the structural prerequisites for the execution of recombination control (DudenhoÈ er et al, 1999) we were able to demonstrate that p53 must be intact within its central DNA-binding and 3' ± 45' exonuclease domain (Vogelstein and Kinzler, 1993;Cho et al, 1994;Mummenbrauer et al, 1996;Janus et al, 1999b) and within its C-terminally neighboring oligomerization domain Wang et al, 1993Wang et al, , 1994Waterman et al, 1995). The extreme C-terminus, executing unspeci®c DNA-binding, reannealing, and strand transfer activities (Oberosler et al, 1993;Brain and Jenkins, 1994;Bakalkin et al, 1994), is dispensable for recombination suppression and 3-stranded junction DNA-binding by p53, but mediates the mismatchdependent stimulation of the basal activity.…”