The presently described culture system may be useful for regenerative medicine, but the primary importance will be as a tool to elucidate the progression of thyroid disease. Moreover, this phenomenon could be induced in vivo because it can be achieved without introducing foreign genes. However, as we have not confirmed full functional ML169 differentiation of the cells, further study is necessary for regenerative application. Nucleotide imbalances, hard to replicate DNA sequences, and damage to the template strand create challenges for complete and accurate DNA replication. The replication stress response maintains genome integrity through sensing and overcoming these challenges by promoting the repair of the damaged DNA, stabilizing stalled replication forks, and activating cell cycle checkpoints. The PI3K-related protein kinases, including ATM and Rad3-related, are primary regulators of the replication stress response. PIKK kinases are large proteins with significant sequence homology and shared domain architecture. The N-terminus of these proteins consist of dozens of Huntington, Elongation factor 3, Protein phosphatase 2A, and PI3K TOR1 repeats; each containing two interacting anti-parallel alpha-helices connected by a flexible loop. The kinase domain is located at the C-terminus and is flanked by the FRAP, ATM, TRRAP domain, the PIKK regulatory domain, and FAT Cterminus domain. The PIKKs preferentially phosphorylate serine or threonine residues followed by a glutamine, giving these kinases many overlapping substrates. PIKK family members promote repair of different types of damaged DNA. Ataxia-telangiectasia mutated is activated by DNA double strand breaks, but ATR signals in response to a variety of DNA lesions, including double strand breaks, base adducts, and crosslinks. The common feature of these lesions is the generation of single stranded DNA either directly or as a consequence of enzymatic processing. Unlike ATM, ATR is essential for the viability of replicating human and mouse cells and is activated every S-phase to regulate replication origin firing, repair stalled replication forks, and prevent early entry into mitosis. Rare, hypomorphic mutations in ATR are associated with Seckel syndrome, a disorder characterized by microcephaly, growth retardation, and other developmental problems. Cancer cells have an increased dependence on the ATR pathway due to high levels of oncogene-induced replication stress and frequent loss of the G1 checkpoint. This dependence makes the ATR pathway a promising cancer therapeutic target. Generation of single stranded DNA gaps initiates ATR activation, which involves recruitment of a signaling complex containing multiple proteins including ATR, ATR-interacting protein, RAD9-HUS1-RAD1, and BRCT repeat protein topoisomerase binding protein 1 to the stalled fork. This recruitment is largely mediated by the single-stranded DNA binding protein, replication protein A. TOPBP1 binds to the ATR-ATRIP complex promoting a conformational change that likely increases its affinity towards substrates. Subcellular localization to specific DNA lesions and additional protein activators are key regulatory elements for the PIKK family members. Additionally, PIKKs are regulated by post-translational modifications. ATM auto-phosphorylation induces the transition from an inactive dimer to an active monomer. Several ATR autophosphorylation sites have been identified, including threonine 1989. However, T1989 is not evolutionarily conserved and there are conflicting data about how important its phosphorylation is to the ATR activation process. Finally, several other proteins have been suggested to regulate ATR activation, but their precise roles may be dependent on the type of initiating signal. In the process of studying how ATR phosphorylation regulates its activity, we discovered that a single mutation at serine 1333 creates a hyperactive kinase. Both the basal activity level and TOPBP1-stimulated activity of the S1333A protein are significantly increased compared to the wild type protein. Additionally, S1333 mutations to glycine, arginine, or lysine also create hyperactive kinases. Conversely, a S1333D mutation decreases ATR activity. While we find no evidence that S1333 is phosphorylated in cultured cells, our studies indicate that mutation of a single serine in the large, HEAT repeat region of this 2,644 amino acid protein is sufficient to greatly alter its activity. The exact mechanism mediating this change will require a highresolution structural analysis; however, these mutants provide useful tools for studying the ATR pathway. Our data indicate that a single amino acid change at position 1333, in a region outside of the known regulatory domains, is sufficient to alter ATR kinase activities. In vitro and in cells, S1333A-ATR is hyperactive compared to wild type ATR while S1333D-ATR is less active. Initially, we hypothesized this amino acid is an auto-phosphorylation site regulating ATR kinase activity. However, we were unable to obtain evidence of phosphorylation in cultured cells or in in vitro kinase reactions. Thus, how the mutations alter kinase activity is not clear, but we hypothesize they alter ATR structure enough to change its ability to bind substrates. S1333 is located within the N-terminal HEAT repeats of ATR. The mechanistic role of the HEAT repeats within PIKK kinases is not known, but HEAT repeats have been shown to serve as protein-protein interaction domains and can also bind DNA. In the structure of DNA-dependent protein kinase, a PIKK family member, the HEAT repeats fold into a double solenoid and form a platform on which the kinase and other C-terminal domains sit. Thus, it is possible that small changes in the HEAT repeat structure are transmitted to the kinase domain, yielding a relatively large and unexpected change in activity. ATRIP also binds to ATR through its HEAT repeats.