At the subcellular level, SOG1 is present in the nucleus, consistent with previous findings; however, unlike any previous data in response to other genotoxins, SOG1 does show a change following Al exposure that is ATR-dependent and may be the result of relocalization, morphological changes to the cell identity, as well as possible diminished visualization due to protein degradation. Post-translational modification of SOG1 was determined to be crucial to the regulation of its function. There is no significant transcriptional change to SOG1 expression in the presence of Al, despite protein accumulation subsiding following Al exposure. This suggests that there may be an undetermined mechanism for protein turnover following SOG1 activation. As the p53 functional homologue in Arabidopsis, SOG1 turnover could prove to be a conserved regulatory mechanism as p53 is ubiquitinated by the E3 ligase, MDM2, in mammals . Perhaps this is even a speculative role for ALT2, as WD40 proteins have been shown to associate with E3 ubiquitin ligases. It is unlikely that ALT2 would be responsible for SOG1 turnover as part of an ubiquitin-proteasome pathway as loss of ALT2 would result in inappropriate persistence of SOG1 and lead to hypersensitivity rather than tolerance as is observed. As of yet, evidence only supports that the activation mechanism in response to Al is dependent on ATR, likely through phosphorylation of SOG1 by ATR. Future studies are needed to determine how SOG1 is degraded following ATR-dependent activation in response to Al as well as to other stresses. As shown in Chapter 5, the fourth Al tolerance gene isolated from the als3-1 suppressor screen was SUV2, nursery pots which encodes the Arabidopsis homologue of the mammalian ATR-interacting protein, ATRIP. The suv2-3 mutation represents a premature stop codon in the eighth exon of SUV2 at amino acid 359 of 646.

After establishing the als3-1 suppressor as an Al tolerant suv2 mutation, it was characterized as being part of the same ATR-mediated pathway, further supporting SUV2 as an interacting partner of ATR in Arabidopsis. Additionally, both cell cycle arrest and differentiation of the QC were established to be dependent on SUV2 following Al exposure, and that these responses ultimately lead to endore duplication as a means to terminal differentiation of the root meristem. Thissue- and cell-specific localization of SUV2 was shown to be present within the cytoplasm and nucleus of actively dividing cells of the root tip. There is accumulation of SUV2 throughout the meristematic regions of the root tip in the absence of Al. When grown in an Al toxic environment, SUV2 persists in the meristematic region of the root tip, but this zone has significantly been reduced in size and therefore there is a concomitant reduction of SUV2 in the root organ. At the sub-cellular level, SUV2 is present in the cytoplasm and the nucleus of cells at the root tip in both the absence and presence of Al. It is likely that as Al causes differentiation of the root meristem up to the point of complete stem cell consumption, the zone of cell division becomes reduced while the zone of elongation encroaches closer to the apex i.e. a morphological reduction in zones of root development. This reduction in the meristematic zone may account for the insignificant but observable progressive reduction in SUV2 transcript levels following treatment with increasing amounts of Al.As with SOG1, SUV2 is phosphorylated by ATR in vitro. While in vivo studies are needed in both cases to confirm this post-translational modification, this would be considered a conserved relationship between ATR and SUV2, as this phosphorylation is known to occur in the homologous proteins in yeast and humans . This begs the question: what are the bona fide substrates of ATR in the Al response pathway? While SOG1 has been demonstrated to have 5 ATM/ATR phosphorylation sites, defined as TQ or SQ motifs, and SUV2 is speculated to contain two sites, in a recent phosphoproteomics study, SOG1 was not even identified as a substrate of ATR or ATM in response to ionizing radiation, let alone SUV2 . Some DNA repair factors such as LIG4, UVH3 and MRE11 as well other DNA maintenance and metabolism factors like CHR4, HTA7/HTA10, PCNA1, MCM4 and H2AXA were proven to be ATR and ATM targets in this kinase target study .

While the authors acknowledge that their experimental technique using adverse tryptic cleavage likely was responsible for not identifying SOG1 in their large-scale identification of kinase targets, this large-scale proteomic endeavor only tested IR stress and did not distinguish between ATR versus ATM phosphorylated targets . Despite the substantial contribution these findings offer to the field of plant DNA maintenance and repair, more in depth studies are needed not just for Al responses, but also for the myriad types of damage to plant DNA that must be repaired. Al treatment leads to root growth inhibition due to terminal differentiation by means of endore duplication as visualized by increases in cell and nucleus size of cells of the root tip. Al treatment results in substantial increases in both cell and nuclear size for als3-1 roots, which is consistent with terminal differentiation in conjunction with endore duplication. This increase in size was shown to be dependent on the Al tolerance factors: ATR, ALT2, SOG1 and SUV2. This shows that all four genetic factors control terminal differentiation and endore duplication in response to Al. Previous studies of a sog1 loss-of-function mutant defined a set of SOG1- mediated genes that were inducible by γ-radiation . In response to Al toxicity, SOG1 and ATR have now been established to be acting within the same response pathway, so it was of great interest to determine if SOG1, ATR, ALT2, and SUV2 were responsible for the induction of this established gene set after treatment with Al. The proper conditions to determine whether Al causes changes in known SOG1-mediates genes was determined by a time course study of the persistence of SOG1 accumulation without completed QC differentiation in response to high Al concentrations. After 4 days of growth on highly inhibitory concentrations of Al, the pool of stem cells in the root meristem differentiate into cells no longer capable of dividing; while SOG1 is completely absent from the root tip after 5 to 6 days of growth on high concentrations of Al. Taken together, this established that 3 days of growth in Al was the optimal point at which expression changes would be analyzed, satisfying the need for SOG1 actively inducing transcriptional changes that would lead to root growth inhibition in response to Al. Genes that were selected for expression analysis were found in a previous study as being highly induced following γ-radiation in a SOG1-dependent manner .

From the γ-radiation study, a number of DNA repair genes like BRCA1, the ortholog for the human breast cancer susceptibility gene; PARP2, a key component of microhomology-mediated DNA repair; GMI1, involved in homologous recombination and chromosome maintenance; and members of the RAD family of genes, RAD17 and RAD51. CYCLINB1;1accumulates in the G2 phase of cell cycle progression and is regulated by transcriptional activation. During normal cell cycle progression, in a population of cells like the root rip, a few would be in the G2 phase at a given time expressing CYCB1;1, but an increase in expression would suggest more cells within the cell population were halted in the G2 phase, indicative of a G2 cell cycle arrest. Other transcription factors were induced in this γ-radiation study,plastic planters like TRFL3 and TRFL10, MYB family transcription factors known to have roles in developmental processes, defense responses and DNA maintenance . In total, 16 genes were assayed . Treatment with Al resulted in a measurable increase in expression of the subset of assayed genes in Col-0 wild type compared with no Al, and for als3-1 there was an even larger increase of gene expression. In contrast, an increase in expression of these genes was not observed for any of the Al tolerant single mutants or double mutants in comparison to the respective controls. This indicates that Al triggers an ATR-, ALT2, SUV2- and SOG1- dependent transcriptional program that is similar to that observed following treatment with γ-radiation, providing an important link between the cell cycle checkpoints involved in DNA damage detection and transcription in response to Al. Clearly, cell cycle checkpoints are emerging as key regulators of Al response, indicating that Al-dependent activation of these factors is central to terminal differentiation following chronic exposure to Al. Unlike γ-radiation, this stoppage of root growth is ATM-independent, as demonstrated by real-time PCR analysis of an atm loss-of-function mutant, indicating that at least in respect to Al stress, SOG1 functions downstream of ATR rather than ATM. This is of particular importance since it is indicative of the type of damage that ATR, ALT2, SOG1 and SUV2 are detecting in the context of Al. There are clear transcriptional differences between Al treatment and γ-radiation. Examples of these differences would be the lack of induction of AtRAD21 in the absence or presence of Al, as well as the ATM independent manner of SOG1-dependent transcript induction in Al treated seedlings while they are ATM-dependent upon exposure to γ-radiation. The difference in the role of ATM in response to these two stresses is interesting, especially considering that ATM is largely uninvolved in the Al response despite the requirement of functional ATR, ALT2, SOG1 and SUV2. This indicates that ATR, ALT2, SOG1 and SUV2 comprise an Al-response pathway that does not primarily require ATM. This suggests that each DNA stress results in a unique transcriptional profile that may be revealing in relation to their respective impacts on genomic integrity. With the overwhelming evidence that the DNA damage response factors ATR, ALT2, SOG1 and SUV2 all play a role in detecting Al dependent damage, it was of interest to examine their roles in response to known genotoxins. HU is an inhibitor of ribonucleotide reductase by scavenging free radicals that are used for the reduction of ribonucleotides . This stalls the replication fork due to depletion of deoxyribonucelotides. ATR functions to detect replication fork blocks and single stranded DNA breaks and atr mutants are sensitive to HU .

MMC is an aziridine containing antibiotic isolated from Streptomyces caepitosus . MMC itself does not react with DNA, but once it becomes reduced by quinone, the aziridine opens and allows MMC to attack the DNA . This reaction forms crosslinks across the complementary strands of the DNA double helix, or interstrand cross links . DNA cross linking can also occur between adjacent bases, called intrastrand DNA cross linking. The chemotherapy drug CDDP is a platinum-containing drug that is a neutral molecule until it is activated through a series of spontaneous aquation reactions, which involve the sequential replacement of the cis-chloro ligands of CDDP with water molecules . When the aquation event occurs, this allows the platinum atom to bind to DNA, preferentially guanine bases, which forms DNA–DNA intrastrand crosslinks . DNA cross links block replication because when they go unrepaired, collapse of the replication fork occurs blocking replication and leading to cell death . ALT2 is the Arabidopsis homologue of the human protein, CSA, which monitors conformational changes in DNA as assessed by blockage ofDNA replication and RNA transcription, a known effect caused by DNA cross links. alt2-1 is hypersensitive to MMC and CDDP . SOG1, the plant functional homologue of the human transcription factor p53, is a transcription factor in Arabidopsis responsible for the initiation of DNA damage responses including DNA repair as well as the initiation of endore duplication in plants. sog1 loss-of-function mutants are sensitive to both the replication fork poison, HU, and the DNA cross linking agents, MMC and CDDP. SUV2 is the Arabidopsis functional homologue to the human protein, ATRIP, which is required for recruiting ATR to sites of DNA damage, presumably to regions coated in Replication Protein A . RPA is a heterotrimeritc protein complex, comprised of sub-units RPA1, RPA2, and RPA3, which binds to single stranded DNA to protect it from nuceolytic degradation and hairpin formation, similar to the prokaryotic Single Stranded Binding protein .