Recombination at chromosomal sequences involved in leukaemogenic rearrangements is differentially regulated by p53

Abstract

Chromosomal translocations and retroviral integration events at breakpoint cluster regions (bcrs) have been associated with leukaemias. To directly compare the effect of different cis-regulatory sequences on recombination, we adapted our SV40 based model system to the analysis of correspondingly selected bcrs from the TAL1, LMO2, retinoic acid receptor alpha (RARalpha) and MLL genes. We show that a 399 bp fragment from the MLL bcr is sufficient to cause a 3-4-fold stimulation of spontaneously occurring DNA exchange and to respond to etoposide by up to 10-fold further elevated frequencies, i.e. to mimic the fragility of the 8.3 kb bcr during chemotherapy. To analyse the regulatory role of p53 in recombination involving leukaemia-related sequences, we stably expressed wtp53 and a transactivation negative mutant. Consistent with the proposed role of p53 as a suppressor of error-prone recombination, both p53 proteins down-regulated recombination with most of the sequences tested, even with the MLL bcr after etoposide treatment. Surprisingly, however, p53 stimulated recombination, in constructs carrying the RARalpha bcr fragment. This is the first study, which provides evidence for a stimulatory role of p53 in homologous recombination. Our data further indicate that inhibition of topoisomerase I can mimic the effects of p53 on stimulating recombination on the RARalpha bcr. Thus, these data also firstly describe a biological role of the biochemical interactions between p53 and topoisomerase I that may have implications for a gain-of-function phenotype of certain p53 mutants in genetic destabilization.

Therapy-related acute myeloid leukemia–like MLL rearrangements are induced by etoposide in primary human CD34+ cells and remain stable after clonal expansion

“…In support of this, the MLL bcr fragment may act in cis to promote spontaneous recombination 3-to 4-fold between simian virus 40 molecules that can be elevated by etoposide treatment. 71 Homology-mediated DSB repair in mammalian cells requires members of the RAD52 epistasis group. 72 Rad51 is central to most homologous recombination events; Rad51 forms nucleoprotein filaments on ssDNA, and mediates homologous pairing and strand exchange between DNA duplexes, 73,74 although single-strand annealing can be promoted by Rad52 in the absence of Rad51.…”

p53 in recombination and repair

“…In the first work, 157 it was discovered that p53 upregulates recombination at a fragment of the RARa breakpoint cluster region, which comprises two perfect topoisomerase 1 recognition sequences. Treatment with the topoisomerase 1 inhibitor camptothecin indicated an epistatic relationship between p53 and topoisomerase 1 with respect to recombination enhancement.…”

“…95 The newly discovered recombination stimulatory activity of p53 that may be connected to topoisomerase 1-dependent excision repair is a good candidate for this gain-of-function phenotype. 157 Thus, failure to suppress aberrant HR coupled to the remaining capacity to stimulate DNA exchange via this topoisomerase 1 repair pathway would be expected to generate a gain-of-function phenotype in recombination, which also could explain the rise in gene amplification observed with some mutant p53 proteins. Specific recruitment and differential modification of the p53 molecule might coordinate the different repair-related activities, as suggested by the tightly regulated interactions of wild-type p53 with topoisomerase 1 in response to DNA damage.…”

Wild-type p53 stimulates homologous recombination upon sequence-specific binding to the ribosomal gene cluster repeat

“…DNA junction binding in particular has been connected to a possible role of wtp53 in the inhibition of gene conversion events (Dudenho¨ffer et al, 1998;Akyu¨z et al, 2002), and, more recently, of replication-associated recombination processes Saintigny and Lopez, 2002). In further support for an antirecombinogenic role of p53 in a DNA sequence-independent manner, p53 was shown to inhibit strand exchange promoted by Rad51 on randomly chosen DNA substrates (Yoon et al, 2004) and to downregulate recombination with different sequences (Boehden et al, 2004). Finally, on the basis of recombination measurements with p53 mutants, it was demonstrated that transcriptional transactivation, which relies on sequence-specific DNA recognition, and consensus sequence-independent downregulation of recombination are genetically separable functions of p53 (Dudenho¨ffer et al, 1999;Saintigny et al, 1999;Willers et al, 2000;Akyu¨z et al, 2002).…”

“…Finally, on the basis of recombination measurements with p53 mutants, it was demonstrated that transcriptional transactivation, which relies on sequence-specific DNA recognition, and consensus sequence-independent downregulation of recombination are genetically separable functions of p53 (Dudenho¨ffer et al, 1999;Saintigny et al, 1999;Willers et al, 2000;Akyu¨z et al, 2002). However, recently published data challenged the notion that p53 regulates homologous recombination in a negative and sequence-independent manner exclusively (Boehden et al, 2004). Thus, when analysing the role of p53 in recombination involving leukemia-related sequences, wtp53 displayed a stimulatory activity directed toward a fragment from the RARa breakpoint cluster region (bcr).…”