Targeted gene repair is a process that corrects single base mutations in genes within the chromosome. Nucleotide alteration is directed by a short single-stranded oligonucleotide, which also serves as a template for the base exchange reaction. The mechanism of action continues to be unraveled but the general system involves a DNA pairing step in which the oligonucleotide aligns to the homologous target site and a correction step in which the genetic information in the oligonucleotide is transferred to the target gene [1,2]. The reaction is catalyzed by enzymes involved in homologous recombination and may be influenced by proteins that regulate the cellular response to DNA damage [3-6]. Damage occurs naturally in mammalian genomes by the action of oxygen radicals, mistakes in DNA synthesis and from exogenous sources such as chemicals or irradiation.
Chemotherapeutic drugs such as VP16 or camptothecin (CPT) or reagents such as hydroxyurea, thymidine, or methyl methanesulfonate (MMS) that cause lesions to accumulate at replication forks [7-10] can be used to induce DNA damage in experimental systems and facilitate studies of gene repair. These lesions inhibit fork movement, leading to a stall in S phase, an event that stimulates gene repair activity. In a recent study, Hu et al. [11] found that the synchronization of cells at the G1/S border with subsequent release enables a higher level of gene repair. The correction levels peak when the majority of cells are in S phase, results which align with earlier data of Majumdar et al. [12]. In addition, Brachman and Kmiec [13,14] demonstrated that DNA replication can influence both the rate and frequency of gene repair. By slowing fork movement, these workers were able to elevate the frequency by activating the homologous recombination (HR) pathway and enabling the correction process to occur with greater efficiency. These data are consistent with earlier reports that had already pointed to HR as a regulatory process of gene repair [4,10]. The induction of HR pathways by DNA damage and the subsequent stimulation of gene repair has provided further clarification of the proteins and pathways involved in the correction events. While earlier findings support a role for DNA damage-induced responses in elevating the frequency of gene repair, little is known about the mechanism of correction i.e. the details of nucleotide exchange. What is known is that regulatory proteins activated by DNA damage events stimulate a cellular response that leads directly or indirectly to a high efficiency of gene repair activity.
The p53 tumor suppressor regulates cell cycle checkpoints, apoptosis, and can transactivate a number of genes involved DNA repair. Furthermore, p53 can act to suppress HR; it is recruited to stalled replication forks to slow or prevent uncontrolled homologous exchanges [15-17]. When p53 was overexpressed in DLD-1 cells simultaneous with the initiation of oligonucleotide-directed gene repair, p53 acts as an antagonist of the reaction, reducing the level of eGFP positive, corrected cells significantly [13]. This suppressive activity could result from p53's role in cell cycle control, DNA damage response, or regulation of senescence and apoptosis. As a long term goal, we seek to modulate and harness the HR repair response as it relates to gene repair activity without inducing the natural suppression associated with the induction of DNA damage.
The amino acid selenomethionine, the major component of Se-enriched foods, has long been demonstrated as a chemopreventative agent, and selenium compounds have yielded encouraging results in current clinical trials to prevent prostate, colon, and other human cancers. Selenomethionine has been reported to induce a DNA repair response and enhance repair-complex formation in treated cells by means of a modulation of p53 activity [18]. The tumor-suppressor function of p53 is usually attributed to its activity at a post-damage stage when it eliminates cells with damaged DNA resulting from genotoxic stress. Selenomethionine seems to induce a different branch of p53 activity, converting p53 into a new conformation through a reduction reaction catalyzed by the protein Ref-1. In this form, p53 does not have any growth-suppressing effect but can effectively modulate DNA repair. Thus, p53 facilitates control over genomic stability by a mechanism that does not require cell death or arrest [18,19]. An agent that modulates gene repair without exhibiting toxicity or inducing DNA damage has numerous advantages, particularly in cases where the level of repair falls below that of clinical relevance and an adjuvant treatment will be required.
Ref-1 is important for the redox regulation of p53, but it is also involved in a number of critical cellular pathways. Ref-1 associates with the human Ape1 protein, forming the multifunctional human AP endonuclease (Ape1/Ref-1). Ape1-Ref-1 functions as an AP endonuclease in DNA repair, and it can activate numerous transcription factors through redox-dependent and redox independent mechanisms [20]. Ref-1 exerts redox control of p53 protein by reducing adjacent cysteine residues (at position 275 and 277 respectively), breaking the disulfide bond and rendering a new p53 conformation. This conformation is associated with increased activation of DNA repair proteins, thus requiring less action for cell cycle arrest or apoptosis.
As described above, selenomethionine has been reported to induce a DNA repair response and enhance repair-complex formation in treated cells. We have reported how to create such a response by inducing a tolerable level of DNA damage that elevates gene repair activity [5,6,21]. In order to advance the technology of gene repair in disease models it is necessary to develop safe and simple methods to increase the efficiency of this repair. Selenium compounds clearly influence DNA damage response pathways [22] often modulating the levels of enzymes known to be involved in gene repair. It should be noted that these authors focused on nucleotide excision repair, a process unrelated to homologous recombination. In any event, we examined the effect of selenium on gene repair activity in a mammalian model cell system in order to gain a greater perspective on the degree of cellular response to the activation of repair pathways.
Answers to all your Biology Questions
