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<p>DNA replication is a fundamental process that must be precisely regulated to ensure timely and faithful transmission of genetic material for proliferation of all organisms. Replication initiation is regulated through a series of precisely timed protein–DNA and protein–protein interactions. In <em>Escherichia coli</em>, one regulatory mechanism of replication initiation occurs through SeqA binding to specific sequences within the <em>oriC</em>, resulting in origin sequestration. SeqA also plays a role in chromosome organization at the replication forks. Despite the functional importance of SeqA in <em>E. coli</em>, its DNA binding mechanism has remained elusive. The work described in this thesis has shown for the first time the minimal functional unit of SeqA that forms a high-affinity complex with DNA through the loss of symmetry. This is a novel observation that explains how SeqA can distinguish template versus newly replicated strand of DNA. We have also identified a protein–protein interaction surface that separates the roles of SeqA at the origin in sequestration and at the replication forks in chromosome organization. The final contribution of the thesis is in the exploration of SeqA functions in other bacterial species and demonstrating the structural and functional similarities between <em>Vibrio cholerae </em>SeqA and <em>E. coli </em>SeqA. Together our work has made a crucial connection between the structural organization of the protein and its functional ability to bind DNA.</p> / Doctor of Philosophy (PhD)

The gene encoding the Dictyostelium replication protein A large subunit (DdRPA1) has been cloned by screening of an EcoR I partial genomic library and a Hind III genomic sub-library. The complete nucleotide sequence, including the promoter region of the gene has been obtained by sequencing. Though the DdRPA1 protein has a size shift during development, 62 kDa in undifferentiated cells and 81 kDa in differentiated cells; they are the products of the same gene. Northern blot analysis revealed that the expression level of the DdRPA1 was constant throughout differentiation and the size of mRNA is the same at all stages, corresponding to a 81 kDa protein. Thus, it seems that the size change between the 62 kDa and 81 kDa is probably due to posttranslational modification, most likely, proteolytic cleavage. The transcription start site for both sizes of DdRPA1 has been identified at 306 bp upstream of the coding sequence by primer extension reaction. A PCR fragment representing 27% of the gene encoding the DdRPA middle size subunit (DdRPA2) has been generated by using the degenerate primers. This PCR fragment has been cloned and sequenced. The mRNA for this subunit corresponds to a protein of about 35 kDa. A decrease of the DdRPA2 mRNA expression level during differentiation was found by comparison between undifferentiated and differentiated cells. In Dictyostelium, replication protein A is a heterotrimeric protein that can bind with specific DNA sequences in a stage-dependent pattern. These DNA sequences were identified as the cis-acting regulatory sites in differentiation-related genes, including the glycogen phosphorylase 2 gene (gp2). Therefore, it is possible that DdRPA is not only a single-stranded DNA binding protein that is used in multiple essential DNA metabolic processes, such as DNA replication, repair and recombination in undifferentiated cells, but also involved in the transcriptional regulation process during differentiation. / Master of Science

Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4’-ethynyl-2’-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (dCK) phosphorylation for its activity and induces replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of dCK bound to EdC and UDP reveals how the rigid 4’-alkyne of EdC fits within the active site of dCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL. / Biomedical Sciences