The product of the tumor suppressor gene, p53, down-regulates the process of homologous recombination [17,31]. The protein may act upon enzymes involved in the DNA damage response pathway or act directly at crossover junctions, effectively stalling their development. Since the process of gene repair requires the activity of proteins involved in HR, we might predict that events that induce HR suppression would likewise result in a reduction in the frequency of gene repair. Previous work in our laboratory [13] and others demonstrated that wild-type p53 can act as an antagonist of the gene repair reaction, whereas overexpression of some mutant p53 proteins exhibit a small yet significant elevation in the frequency of gene repair [13]. The suppressive action of wild-type p53 [17] may affect the gene repair reaction through the binding to replication forks which have been shown to be a key intermediate (and in fact a stimulant) in the correction process [26].
We observe that the overexpression of wild-type p53 gene in HCT116 leads to a reduction in gene repair, confirming data from Pierce et al. [32] and Brachman et al. [13]. The study by Pierce et al. utilized cell-free extracts as sources of gene repair activity while Brachman and Kmiec tested p53 overexpression directly in cells [13]. The inhibitory effect of p53 in DLD-1 cells which contain both mutant and wild-type alleles was clearly evident. Mutations in the DNA binding domain of the p53 gene can cause the p53 protein to lose the ability to suppress homologous recombination [31,33,34]. Mutant forms of p53 may not be able to inhibit Rad51-mediated strand exchange or the reverse branch migration of stalled replication forks [17,35]. Expression of several mutant p53 proteins, specifically, the p53 175H mutant, which inhibits G1 checkpoint control, can prevent the suppression of HR and stimulate replication inhibition-induced HR [9,15]. Here, we utilize HCT116 cells that have a normal genetic complement of wild-type p53 genes and show that additional p53 suppresses the frequency of gene repair even further.
Our data suggest that suppression of gene repair activity most likely does not occur through an elevation in p53 expression nor by the activation of a well-known p53 conformer. Thus, we explored a novel explanation for the suppressive nature of selenomethionine in the gene repair reaction [20,36], showing that the addition of selenomethionine (SeMet) to HCT116 cells leads to a reduction in gene repair activity. Selenium compounds have been shown to elevate p53 activity [37,38] and to stimulate DNA damage response pathways through a phosphorylation cascade that includes ATM and H2AX [22]. Gene repair activity has been shown to be sensitive to the activities of proteins involved in the response to DNA damage [13,14]. Thus, it was of interest to determine which selenium-induced pathway would predominate in this system; would increased DNA repair activities promote correction or would induction of p53 lead to suppression. We observed that gene repair is possibly inhibited by a pathway involving p53. This pathway includes Ref-1 which controls the activity of p53 through redox activation [20]. Ref-1 is also a key protein in the activation of other proteins including transcription factors and enzymes involved in nucleotide excision repair [20]. While the downstream effects of Ref-1 induction are numerous, we focused on the activation of the Ref-1 protein by SeMet. We show that under conditions that promote gene repair, SeMet induces Ref-1 expression with a concurrent diminution of correction. The reduction by SeMet aligns with the effect of p53 overexpression (Figure 4A and 4B) and the two appear to act synergistically. Taken together, these data suggest that inhibition of gene repair by SeMet most likely takes place in a pathway involving Ref-1. The most likely downstream target for Ref-1, especially in response to DNA damage is p53. Thus, consistent with the previous reports, we believe that p53 plays a critical, if not suppressive role, in regulating the frequency of gene repair.
Our data are consistent with a role for homologous recombination in the mechanism of gene repair [26]. A number of key proteins are involved in modulating the frequency of correction. These include Rad51, Rad52 and Rad57 [39] as well as ATM, CHK1 and CHK2 [40]. Based on the data presented herein, we can conclusively add p53 to that list. This protein can inhibit homologous recombination functions by binding to the three-stranded recombination intermediate and destabilizing it [35,41]. As shown by Drury et al. [42], the three-stranded intermediate is a requisite structure in the gene repair pathway and thus one can envision that p53 suppression activity occurs through the destabilization of structural intermediates. Alternatively, the activation of p53 by selenomethionine, mediated by Ref-1, could inhibit the progression of replication forks, by enabling fork regression [17,43]. This reversal reduces the number of targets for gene repair because correction events rely heavily on the process of DNA replication [26]. Thus, induction of p53 activity will remain a barrier to the successful application of gene repair as therapy for genetic disorders.
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