p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches

Abstract

Insertion/deletion (IDL) mismatches in DNA are lesions consisting of extra bases on one strand. Here, the binding of p53 and its 14 kDa C-terminal domain to DNAs containing one or three 3-cytosine IDL mismatches was examined. Electron microscopy showed that both p53 forms bound predominantly as tetramers at the lesions while single-stranded binding proteins did not bind. Gel retardation assays showed that p53 formed highly stable complexes when the DNA contained the IDL mismatches, but only unstable complexes when the DNA lacked lesions (but did contain free ends). The highly stable complexes had a half-life of > 2 hr, suggesting that upon encountering lesions, p53 may recruit other proteins to the site, providing a signal for DNA damage.

Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation

“…Our present data would suggest that certain MTp53 proteins may confer a`gain of function' in this development of the radioresistant phenotype, and is supported by other experiments in our laboratories which have shown increased radioresistance among null-p53 transformed cell lines transfected with a MTp53 allele (Bristow et al, manuscript in preparation). Recently, it has been hypothesized that the stabilization of p53 proteins observed following DNA damage may represent the formation of complexes between the carboxy-termini of p53 proteins and sites of DNA damage, perhaps signalling the recruitment of multi-protein repair complexes (Jayaraman and Prives, 1995;Lee et al, 1995). Of note, the p53 carboxy-terminus is rarely a site for p53 gene mutation, and missense-MTp53 proteins retain the ability for non-speci®c DNA binding and DNA strand re-annealing following DNA damage (Bakalkin et al, 1995;Reed et al, 1995;Wu et al, 1995).…”

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

“…Strikingly, p53(1 ± 320) had completely lost the ability to recognize these 3-stranded DNA-structures at concentrations, at which wild-type p53 tetramers and octamers fully titrated out the substrate, and at which also p53(1 ± 360) performed strong binding (see Figure 3c). For a more detailed analysis of p53(1 ± 360), which is devoid of the amino acid stretch implicated in DNA damage recognition (Jayaraman and Prives, 1995;Lee et al, 1995;Reed et al, 1995), we titrated DNA substrates for a nity measurements with 3-stranded junctions containing perfectly matching doublestranded termini versus junctions comprising an A-G mismatch. Intriguingly, the deletion of the C-terminal 30 amino acids resulted in practically identical K Dvalues for both substrates (Table 3), whereas wild-type p53 displays comparable high-a nity binding only with the speci®c mismatch substrate (DudenhoÈ er et al, 1998).…”

“…These data indicated that biochemical functions of p53 attributed to the C-terminal 30 amino acids are unlikely candidates for the mechanism underlying the suppression of recombination events. Consequently, neither binding or reannealing of single-stranded DNA ends within recombination intermediates (Oberosler et al, 1993;Brain and Jenkins, 1994;Bakalkin et al, 1994) nor recognition of DNA damage (Jayaraman and Prives, 1995;Lee et al, 1995;Reed et al, 1995;Wu et al, 1995) seemed to be essential for this process. However, we were able to attribute the recognition of speci®c mismatches on recombination intermediates to this short domain of p53.…”

“…The C-terminal 30 amino acids play a crucial role in the recognition of several DNA structures related to DNA damage (dsDNA ends, ssDNA, insertion/deletion mismatches) and are essential for RNA and DNA reannealing activities (Oberosler et al, 1993;Brain and Jenkins, 1994;Bakalkin et al, 1994;Jayaraman and Prives, 1995;Lee et al, 1997;Reed et al, 1995;Wu et al, 1995). In its native state, this region negatively controls sequence-speci®c DNA binding of the core, but interactions with co-factors, DNA damage binding, and post-translational modi®cations can neutralize this e ect (Jayaraman and Prives, 1995;Lee et al, 1995;Bayle et al, 1995;Reed et al, 1995;Selivanova et al, 1996;Hupp et al, 1992). p53 lacking the C-terminus is constitutively active for transactivating transcription, a situation resulting in vivo in a species-and tissuespeci®c manner from alternate splicing (Wolf et al, 1985;Kulesz-Martin et al, 1994), or by autoproteolytic cleavage after sensing damaged DNA (Okorokov et al, 1997).…”

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments

“…Wild-type (wt) p53 acts as a tumour suppressor gene product (Finlay et al, 1989), inhibiting the proliferation of tumour cells in culture and suppressing the tumourigenicity of tumour cells in nude mice (Baker et al, 1990;Mercer et al, 1990). It is clearly involved in the cell cycle arrest resulting of a genomic stress (UV irradiation or chemotherapeutic agent), in DNA repair and apoptosis (Kastan et al, 1991;Clarke et al, 1993;Lowe et al, 1993;Lu and Lane, 1993;Zhan et al, 1993;Eizenberg et al, 1995;Guillouf et al, 1995;Lee et al, 1995;Smith et al, 1995). Wt p53 is a sequence-speci®c transcription factor, which regulates the expression of genes involved in growth control and apoptosis, such as p21/WAF1/CIP1 (E1-Deiry et al, 1993), GADD45 (Kastan et al, 1992), IGF-BP3 (Buckbinder et al, 1995), Bax (Miyashita et al, 1995), FAS/APO1 (OwenShaub et al, 1995) and more recently, genes involved in free radical formation (Polyak et al, 1997;see Ko and Prives, 1996;Levine, 1997, for review).…”