DNA double-strand breaks (DSBs) are highly cytolethal DNA lesions. To protect genomic integrity and ensure cellular homeostasis, cells initiate a complex signaling-based response that activates cell cycle checkpoints, coordinates DNA repair, regulates gene expression and, if necessary, induces apoptosis. The spatio-temporal control of this signaling pathway relies on a large number of post-translational modifications, including phosphorylation and regulatory ubiquitylation. In this thesis, I describe the discovery and characterization of the E3 ubiquitin ligase RNF168, which cooperates with the upstream E3 ubiquitin ligase RNF8 to form a cascade of regulatory ubiquitylation at damaged chromatin. One of the main functions of RNF8/RNF168-dependent chromatin ubiquitylation is to generate a molecular landing platform for the ubiquitin-dependent accumulation of checkpoint and DNA repair proteins such as 53BP1, the breast-cancer associated protein BRCA1 and the RNF168-paralog RNF169. I present evidence that the hierarchical recruitment of these proteins to DSB sites is, in large part, organized through the use of tandem protein interaction modules. These modules are composed of a ubiquitin-binding domain and an adjacent targeting motif called LRM, which specifies the recognition of RNF8- and RNF168-ubiquitylation substrates at damaged chromatin. I conclude that the LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the chromatin-based response to DSBs. Collectively, my results indicate that RNF168-mediated chromatin ubiquitylation is critical for the physiological response to DSBs in human cells. The importance of the ubiquitin-based response to DSBs is underscored by the finding that RIDDLE syndrome, an immunodeficiency and radiosensitivity disorder, is caused by mutations in the RNF168 gene.

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The budding yeast Saccharomyces cerevisiae exhibits exquisite control of cellular size in response to the nutritional composition of its environment. Size control is mediated at the G1/S phase transition, termed Start: passage through Start represents an irreversible commitment to cell division and is contingent on achieving a critical size. When nutrients are plentiful, yeast increase their critical size set-point resulting in larger cells; in contrast, in poor nutrients, yeast pass Start at a smaller size. The genetic basis for nutrient-dependent size control and the means by which yeast sense their size remain elusive. One measure of growth potential is ribosome biogenesis, the rate of which correlates with cell size. I characterized a G-patch domain containing protein, Pfa1, which has been shown to activate the helicase activity of the pre-rRNA processing factor Prp43. Intriguingly, Pfa1 is multiply phosphorylated in response to inhibition of the TOR kinase, the central player in growth regulation. This phosphorylation occurs in a region required for Pfa1 function in ribosome biogenesis, independent of its role as a helicase activator. Consistently, phosphorylation correlates with loss of physical interactions with ribosome biogenesis and altered interactions with the ribosome. Mutation of these phosphorylation sites eliminates TOR-dependent phospho-regulation, and confers sensitivity to TOR inhibition. I propose a model wherein Pfa1 is phosphorylated in response to nutrient stress, leading to relocalization of essential processing factors, and inhibition of both ribosome biogenesis and tRNA maturation. Further, I constructed and verified a non-covalent short oligonucleotide barcode microarray platform, and applied it to genome-scale parallel analyses of both the DNA damage response and cell size control in S. cerevisiae. Through these studies, I uncovered novel connections between size control and numerous cellular processes including: the large subunit of the ribosome; the mitochondrial pH gradient; and proteins involved in oxidant-induced cell cycle arrest.

The budding yeast Saccharomyces cerevisiae exhibits exquisite control of cellular size in response to the nutritional composition of its environment. Size control is mediated at the G1/S phase transition, termed Start: passage through Start represents an irreversible commitment to cell division and is contingent on achieving a critical size. When nutrients are plentiful, yeast increase their critical size set-point resulting in larger cells; in contrast, in poor nutrients, yeast pass Start at a smaller size. The genetic basis for nutrient-dependent size control and the means by which yeast sense their size remain elusive. One measure of growth potential is ribosome biogenesis, the rate of which correlates with cell size. I characterized a G-patch domain containing protein, Pfa1, which has been shown to activate the helicase activity of the pre-rRNA processing factor Prp43. Intriguingly, Pfa1 is multiply phosphorylated in response to inhibition of the TOR kinase, the central player in growth regulation. This phosphorylation occurs in a region required for Pfa1 function in ribosome biogenesis, independent of its role as a helicase activator. Consistently, phosphorylation correlates with loss of physical interactions with ribosome biogenesis and altered interactions with the ribosome. Mutation of these phosphorylation sites eliminates TOR-dependent phospho-regulation, and confers sensitivity to TOR inhibition. I propose a model wherein Pfa1 is phosphorylated in response to nutrient stress, leading to relocalization of essential processing factors, and inhibition of both ribosome biogenesis and tRNA maturation. Further, I constructed and verified a non-covalent short oligonucleotide barcode microarray platform, and applied it to genome-scale parallel analyses of both the DNA damage response and cell size control in S. cerevisiae. Through these studies, I uncovered novel connections between size control and numerous cellular processes including: the large subunit of the ribosome; the mitochondrial pH gradient; and proteins involved in oxidant-induced cell cycle arrest.

hCLCA2 and hCLCA4 are chloride channel regulators that are expressed in normal breast epithelial cells and frequently downregulated in breast cancers. Recent investigations revealed that these two proteins may have a role in suppressing breast cancer progression. In this thesis, I will address their role in maintaining epithelial differentiating and inhibiting cell proliferation of breast epithelial cells. The epithelial to mesenchymal transition (EMT) is a developmental program in which epithelial cells downregulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In breast cancer, EMT facilitates invasion of surrounding tissues and correlates closely with cancer metastasis and relapse. We found previously that the candidate tumor suppressor hCLCA2 is a p53-inducible proliferation-inhibitor that is frequently lost in breast cancer. We show here that another member of the CLCA gene family, hCLCA4, is expressed in mammary epithelial cells and is similarly downregulated in breast tumors and in breast cancer cell lines. Like CLCA2, the gene is stress-inducible, and ectopic expression inhibits colony formation. Transcriptional profiling studies revealed that hCLCA4 and hCLCA2 together are markers for mammary epithelial differentiation, and both are downregulated by TGF beta. Moreover, knockdown of either on in immortalized cells by shRNAs caused downregulation of epithelial marker E-cadherin, while mesenchymal markers N-cadherin, vimentin, and fibronectin were upregulated, indicating an EMT program. Double knockdown of hCLCA2 and hCLCA4 enhanced the mesenchymal profile. These findings suggest that hCLCA4 and hCLCA2 play complementary but distinct roles in epithelial differentiation. Clinically, low expression of hCLCA2 and hCLCA4 signaled lower relapse-free survival in breast cancers. Cellular senescence is a program of irreversible cell cycle arrest in response to stressors such as DNA damage, ROS, telomere erosion, or oncogene activation. It is one of the primary tumor suppression mechanisms mediated by p53 and is often disabled in cancer cells. However, the downstream signaling pathway whereby p53 induces cellular senescence remains incomplete. We reported previously that hCLCA2 was a p53 inducible gene that is downregulated with breast cancer progression. We and other group noticed that hCLCA2 was induced in parallel with several types of senescence. Lentiviral transduction of CLCA2 into MCF7 cells inhibited cell proliferation and cells showed senescence phenotype. To investigate the mechanism biochemically, we used pAd-Easy to express hCLCA2 in the model breast cancer cell line CA1d. A protein expression profile of these cells over a 6 day period revealed induction of p21, p53, and the DNA damage-response pathway. To test whether hCLCA2 is required for the cellular senescence process, hCLCA2 was knocked down in HMLE. The knockdown cells (KD) and negative control were treated with a low concentration of doxorubicin, and cell proliferation was measured. The KD cells were more resistant to growth inhibition by doxorubicin. Moreover, a time course experiment showed that induction of SA beta-galactosidase, DNA damage response, and lysosomal markers IFI30 and CTSS was delayed in the knockdown cells. These results suggest that hCLCA2 plays an important role in DNA damage response and the senescence program.

Modular domains of proteins are important in cellular signaling processes. Eukaryotic cells are constantly undergoing DNA damage due to exogenous and endogenous sources of damage. The DNA damage response (DDR) involves a complex network of signaling events mediated by modular domains such as the BRCT (BRCA1 C-terminal) domains. Therefore, proteins containing BRCT domains are important for DNA damage detection and signaling. In this dissertation, we focus on two BRCT-containing proteins BRCA1 and PAXIP1. BRCA1 is a gene that is known to be associated with increased risk of hereditary breast and ovarian cancer. Germline variants of BRCA1 are assessed to determine lifetime risk of developing breast and ovarian cancer. This is performed by genetic testing of the BRCA1 sequence and the variants can be classified as pathogenic, non-pathogenic or variants of unknown significance (VUS). Using family history, segregation analysis, co-occurrence and tumor pathology, certain variants have been classified as either pathogenic or non-pathogenic. However, a large majority of the variants are classified as VUS. Functional assays are critical in providing insight in the case of VUS results. We have a developed a visualization resource to aid in functional analysis of BRCA1 missense variants that occur due to single amino acid changes. This tool is known as BRCA1 Circos (http://research.nhgri.nih.gov/bic/circos/) and it aggregates, harmonizes and allows interpretation of data from all published studies on functional analysis of BRCA1 missense variants. Therefore, this is an important tool that will aid in the meta-analysis of functional data needed to better assess VUS. Functional studies of BRCA1 also demonstrate that majority of the variants that have a functional impact on the protein lie in the BRCT region of the protein. This indicates that the BRCT region is important in cancer development. To further analyze the function of BRCT-containing proteins, a study was previously undertaken to evaluate the role of BRCT-containing proteins and their interaction partners in the DNA damage response and consequently, cancer. BRCT domains of seven BRCT-containing proteins were used as baits and their binding partners were demonstrated to be highly enriched in the DDR process. We hypothesized that members of this BRCT-centric protein-protein interaction network could constitute targets for sensitization to DNA damaging chemotherapy agents in lung cancer. Therefore, we probed this established dataset containing the protein-protein interaction network (PPIN) of seven BRCT-containing proteins to identify seventeen kinases. A systematic pharmacological screen was performed to evaluate these kinases as targets to enhance platinum-based chemotherapy in lung cancer and this revealed WEE1, a mitotic kinase, as a potential target. Of the seventeen kinases, inhibition of mitotic kinase, WEE1, was found to have the most effective response in combination with platinum-based compounds in lung cancer cell lines. In the PPIN, WEE1 was shown to interact with PAXIP1 (PTIP), a BRCT-containing protein involved in transcription and in the cellular response to DNA damage. PAXIP1 has been shown to bind DDR proteins, such as 53BP1 and γH2AX, and also shown to be an important part of immune development. In this dissertation, we observe that WEE1 binds to PAXIP1 and PAXIP1 regulates the WEE1-mediated phosphorylation of its main substrate, CDK1. We also demonstrate that ectopic expression of PAXIP1 combined with WEE1 inhibitor, AZD1775, leads to an increase in the mitotic index at the G2/M checkpoint. Overexpression of PAXIP1 combined with AZD1775 treatment in cells with prior DNA damage causes high levels of caspase-3 mediated apoptosis as compared to AZD1775 treatment alone. In summary, we identify the role of PAXIP1 in sensitizing lung cancer cells to the WEE1 inhibitor, AZD1775, in combination with platinum-based therapy and propose the use of WEE1 and PAXIP1 levels as mechanism-based biomarkers. Overall, these studies indicate that BRCT-containing proteins through their role in the DDR and the cell cycle are crucial for both cancer prevention and therapy.

Abstract Genetic information is stored in the base sequence of DNA. As DNA is often damaged by radiation or reactive chemicals, cells have developed mechanisms to correct the DNA lesions. These mechanisms involve recognition of damage, DNA repair and cell cycle delay until DNA is restored. Failures in the proper processing of DNA lesions may lead to mutations, premature aging, or diseases such as cancer. In this thesis study the human topoisomerase IIβ binding protein 1 (TopBP1) was identified as the homolog of budding yeast Dpb11 and fission yeast Cut5. TopBP1 was found to be necessary for DNA replication and to associate with replicative DNA polymerase ε. TopBP1 localised to the sites of DNA damage and stalled replication forks, which suggests a role in the DNA damage response. TopBP1 interacted with the checkpoint protein Rad9, which is a part of a protein complex whose function includes tethering proteins to sites of DNA damage. This supports a role for TopBP1 in the early steps of checkpoint activation after DNA damage. TopBP1 also interacted with the tumour suppressor protein p53 in a phosphorylation dependent manner. In addition, the data support a role for TopBP1 outside of S-phase. During M-phase, TopBP1 was found to localise to centrosomes along with the tumour suppressor proteins Brca1 and p53. Analysis of the expression of TopBP1 in mouse tissues suggested that TopBP1 may also play a role during meiosis. The localisation pattern of TopBP1 in mouse meiotic spermatocytes resembled that of many proteins functioning during meiotic recombination. For example, co-localisation of ATR kinase and TopBP1 was observed during meiotic prophase I. In accordance with the findings from mouse studies, the analysis of a cut5 mutant during yeast meiosis showed that Cut5 is essential for the meiotic checkpoint. These results strongly suggest that TopBP1 operates in replication and has checkpoint functions during both the mitotic and meiotic cell cycles.

Abstract DNA polymerase ε from HeLa cells has been purified as a heterodimer of a 261 kDa catalytic subunit and a tightly associated smaller polypeptide, the B-subunit. The cDNAs encoding the B-subunits of both human and mouse Pol ε were cloned and shown to encode proteins with a predicted molecular weight of 59 kDa. These subunits are 90 % identical and share 22 % identity with the 80 kDa B-subunit of Saccharomyces cerevisiae Pol ε. The gene for the human Pol ε B-subunit was localized to chromosome 14q21-q22 by fluorescence in situ hybridization. Primary structure analysis of the Pol ε B-subunits demonstrated that they are similar to the B-subunits of Pol α, Pol δ and archaeal DNA polymerases, and comprise a novel protein family of DNA polymerase associated-B-subunits. The family members have 12 conserved motifs distributed in the C-terminal parts, which apparently form crucial structural and functional sites. Secondary structure predictions indicate that the B-subunits share a similar fold, and phylogenetic analysis demonstrated that the B-subunits of Pol α and ε form one subfamily, while the B-subunits of Pol δ and the archaeal proteins form a second subfamily. The corresponding eukaryotic and archaeal catalytic subunits are not related, but all have the characteristics of replicative DNA polymerases. This indicates that the B-subunits of replicative DNA polymerases from archaea to eukaryotes belong to the same protein family and perform similar functions. In S. cerevisiae, Pol ε associates with the checkpoint protein Dpb11. In this study, a human protein, TopBP1, with structural similarity to the budding yeast Dpb11, fission yeast Cut5 and the breast cancer susceptibility gene product Brca1 was identified. The human TOPBP1 gene localizes to chromosome 3q21-q23 and encodes a phosphoprotein of 180 kDa. TopBP1 has eight BRCT domains and is also closely related to the recently identified Drosophila melanogaster Mus101. TopBP1 expression is induced at the G1/S boundary and it performs an important role in DNA replication, as evidenced by inhibition of DNA synthesis by TopBP1 antiserum in isolated nuclei. TopBP1 also associates with Pol ε and localizes, together with Brca1 to distinct foci in S-phase, but not to sites of ongoing DNA replication. Inhibition of DNA replication leads to re-localization of TopBP1 and Brca1 to stalled replication forks. DNA damage induces formation of distinct TopBP1 foci that co-localize with Brca1 in S-phase, but not in G1-phase. The role of TopBP1 in the DNA damage response is also supported by the interaction between TopBP1 and the human checkpoint protein hRad9. These results implicate TopBP1 in replication and checkpoint functions.

During my PhD, I have investigated aspects of the DNA damage response (DDR) in the context of three different cellular scenarios: DNA damage signalling in response to double-strand breaks during mitosis, coordination of DNA replication with DNA damage responses by regulation of the GINS complex, and checkpoint activation by the prototypical checkpoint protein Rad9. Here, I show that mitotic cells treated with DNA break-inducing agents activate a 'primary' DDR, including ATM and DNA-PK-dependent H2AX phosphorylation and recruitment of MDC1 and the MRN complex to damage sites. However, downstream DDR events and induction of a DNA damage checkpoint are inhibited in mitosis, with full DDR activation only ensuing when damaged mitotic cells enter G1. In addition, I provide evidence that induction of a primary DDR in mitosis is biologically important for cell viability. The GINS complex is an evolutionarily conserved component of the DNA replication machinery and may represent an ideal candidate for transferring the DNA damage signal to the replication apparatus. Here, I show the identification of a consensus 'SQ' PIKK phosphorylation motif at the carboxyl end of the GINS complex subunit, Psf1. In Saccharomyces cerevisiae, switching the conserved serine to a glutamic acid is lethal, indicating that the site is crucial for the protein's function. Moreover, in human cells, I identified UV-DDB, a heterodimeric complex involved in NER repair, as a binding partner that specifically interacts with the Psf1 C-terminus in vitro. Finally, I discuss my findings in characterizing functional interactions between Rad9 and Chk1 in S. cerevisiae. I show that specific consensus CDK sites within Rad9 N-terminus are essential to enable Chk1 phosphorylation and activation, and that MCPH1, a human homologue of Rad9, may share a conserved function in binding and activating Chk1, underscoring the evolutionarily conservation of checkpoint activation mechanisms.