In vivo assay of p53 function in homologous recombination between simian virus 40 chromosomes

Abstract

To investigate a possible role of p53 in DNA exchange mechanisms, we have developed a model system which allows us to quantify homologous recombination rates in eukaryotic cells. We generated two types of simian virus 40 (SV40) whose genomes were mutated in such a way that upon double infection of monkey cells, virus particles can be released only after interchromosomal exchange of genetic material. This test system allowed us to determine recombination rates in the order of 10 ؊4 to 10 ؊6 for chromatin-associated SV40 genomes. To study the role of p53-T-antigen (T-Ag) complexes in this process, we designed viral test genomes with an additional mutation leading to a single amino acid exchange in T-Ag (D402H) and specifically blocking T-Ag-p53 interactions. Analysis of primary rhesus monkey cells endogenously expressing wild-type p53 showed a decreased recombination rate upon loss of efficient T-Ag-p53 complex formation. However, cells expressing mutant p53 (LLC-MK 2 cells), the introduction of mutant T-Ag did not affect the DNA exchange rates. Our data are interpreted to indicate an inhibitory role of wild-type p53 in recombination. In agreement with this hypothesis, p53-T-Ag complex formation alleviates the inhibitory effect of wild-type p53.

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

“…This conclusion is drawn from our finding that specifically the bound, and not the unbound, substrate was degraded upon addition of Mg 2ϩ ions as a cofactor, starting the p53 exonuclease activity. Regarding the biological relevance of this interaction, it may be more than a coincidence that binding of SV40 T-Ag to p53 abolished the p53 exonuclease activity in vitro (this study) and enhanced the frequency of recombination in SV40-infected cells by at least 1 order of magnitude (68). However, more direct proof for an in vivo involvement of the p53 exonuclease activity in recombination or other repair events has to be obtained in order to substantiate our model of a dual role for p53 in maintaining genomic integrity (25).…”

“…To get further clues about a possible in vivo function of the p53 exonuclease, we here followed up our previous observations that p53 negatively regulates homologous recombination, possibly by controlling fidelity of homologous recombination via specific mismatch recognition (8,68). wt p53 specifically binds to three-stranded DNA substrates mimicking early recombination intermediates and rapidly and efficiently degrades the bound substrate when the exonuclease activity of p53 is switched on by addition of Mg 2ϩ as a cofactor.…”

p53's double life: transactivation-independent repression of homologous recombination

“…Two main methods have been used to measure HR. The first method measures the intermolecular HR between two defective SV40 genomes with HR recreating a functional viral genome [11]. The second strategy measures intramolecular HR between tandem repeat sequences, which restores a functional reporter gene [12 -18].…”

Adenovirus type 12 early region 1B 54K protein significantly extends the life span of normal mammalian cells in culture

“…Such instability of telomeric DNA may explain one of the effects of the loss of wild-type p53 activity, i.e., the genomic instability and/or chromosome loss that is observed in the absence of functional p53 (36,51). In support of this hypothesis, it has recently been shown that there is an inhibitory effect of p53 on homologous recombination of SV40 chromosomes (69). In addition, p53 has been implicated in the regulation of centrosome duplication (23), and therefore the transcriptional regulatory functions of p53 may not be uniquely important to cell cycle control and life span regulation.…”