Different Regulation of the p53 Core Domain Activities 3′-to-5′ Exonuclease and Sequence-Specific DNA Binding

Abstract

In this study we further characterized the 3'-5' exonuclease activity intrinsic to wild-type p53. We showed that this activity, like sequence-specific DNA binding, is mediated by the p53 core domain. Truncation of the C-terminal 30 amino acids of the p53 molecule enhanced the p53 exonuclease activity by at least 10-fold, indicating that this activity, like sequence-specific DNA binding, is negatively regulated by the C-terminal basic regulatory domain of p53. However, treatments which activated sequence-specific DNA binding of p53, like binding of the monoclonal antibody PAb421, which recognizes a C-terminal epitope on p53, or a higher phosphorylation status, strongly inhibited the p53 exonuclease activity. This suggests that at least on full-length p53, sequence-specific DNA binding and exonuclease activities are subject to different and seemingly opposing regulatory mechanisms. Following up the recent discovery in our laboratory that p53 recognizes and binds with high affinity to three-stranded DNA substrates mimicking early recombination intermediates (C. Dudenhoeffer, G. Rohaly, K. Will, W. Deppert, and L. Wiesmueller, Mol. Cell. Biol. 18:5332-5342), we asked whether such substrates might be degraded by the p53 exonuclease. Addition of Mg2+ ions to the binding assay indeed started the p53 exonuclease and promoted rapid degradation of the bound, but not of the unbound, substrate, indicating that specifically recognized targets can be subjected to exonucleolytic degradation by p53 under defined conditions.

Dissociation of p53-mediated suppression of homologous recombination from G1/S cell cycle checkpoint control

“…StuÈ rzbecher et al (1996) mapped the interaction with the Rad51 recombinase, a central homologous pairing and strand exchange protein, to central regions of the p53 protein. The central core domain was found to contain p53's intrinsic exonuclease activity (Mummenbrauer et al, 1996;Janus et al, 1999b) and also binds non-speci®cally to internal regions of single-stranded DNA (Bakalkin et al, 1994). Apparently, the conformational change induced by the amino acid exchange Alanin to Valin at position 135 was not su cient to disrupt the presumptive regulatory domain in our study.…”

“…Consistent with these data, p53 was recently observed to bind speci®cally to Holliday junctions (Lee et al, 1997). Therefore, one way of directly regulating HR could involve a mismatch repair activity of p53 (DuddenhoÈ er et al, 1998) which may be related to its recently demonstrated 3' to 5' exonuclease activity (Mummenbrauer et al, 1996;Janus et al, 1999b) and which would parallel the described anti-recombinogenic properties of the MutSHL mismatch repair system (Fishel and Kolodner, 1995).…”

“…Furthermore, p53 has been suggested to act in a post-replicative mismatch repair pathway (Mummenbrauer et al, 1996;Huang, 1998), it co-localizes with DNA synthesis and the DNA replication apparatus (Huang, 1998), interacts with viral replication (Deppert, 1994), migrates into the nucleus with S phase (Shaulsky et al, 1990;Martinez et al, 1991), and interacts with a variety of proteins involved in DNA repair (for further review, see Janus et al, 1999a). Deppert and colleagues (Janus et al, 1999b) recently made the intriguing observation that p53's exonuclease and sequencespeci®c DNA binding activities appeared to be mutually exclusive functions. This has led the authors to formulate a dual p53 model which may signi®cantly extend p53's role as a guardian of the genome (Janus et al, 1999a), and which is supported by our data: In addition to the well documented cellular stress responses mediated by activated p53, the model postulates regulatory functions in DNA metabolism such as repair of endogenous DNA damage, control of replication and recombination when p53 is in its latent' state which is generally considered inactive for transcriptional regulation.…”

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

“…Our mapping data might be interpreted to exclude a major contribution of p53's exonuclease activity in controlling recombination, as the same C-terminal truncation in p53(1 ± 333), which causes the loss of recombination control, results in a stimulation of exonucleolytic degradation by more than one order of magnitude (Janus et al, 1999). We noticed slightly elevated recombination rates for LLC-MK 2 [p53(1 ± 333)her] cells, which might be the consequence of unrestrained nucleolytic processing by p53(1 ± 333)her.…”

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

“…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.…”