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DNA replication and associated processes take place in all living organisms with the same constitutions. The knowledge of the duplication process, chromatin building and repair mechanisms has increased explosively over the last years, but the complex interplay of different proteins and their mechanisms are not conceived properly. During DNA replication, the DNA has to be unpacked, duplicated and finally repacked into chromatin. These steps require different proteins, e.g. new histone proteins on demand to secure an error-free and undelayed DNA replication. This thesis includes different mathematical models for DNA replication, repair and chromatin formation, which are based on experimental results. Three models of chromatin formation provide a simplified description of histone gene expression and protein synthesis during G1/S/G2 phase and include the contribution of different regulatory elements. Furthermore, all models present two different mechanisms of regulation to test possible scenarios of newly synthesised histones and free DNA binding sites. The basic model presents a single histone gene, which codes for a single histone protein. The stem-loop binding protein (SLBP) acts as a master regulator, which is only present during S phase. Different analyses of early S-phase, over- and underexpressed replication and the down-regulation of SLBP proof the model under extreme conditions. This basic model serves as a template for further scenarios with several genes and different histone families. For this, a second model is realised to simulate imbalances in the histone mRNA synthesis and translation. Additionally, a third model tests a gene knock-out and mRNA silencing. The initial histone model is able to qualitatively reproduce experimental observations and shows basic regulatory principles. The adaptation with several genes and different histone families presents qualitatively different system responses for the discussed regulatory mechanisms and illustrates the ability to compensate the effect of mRNA silencing.

Highly active antiretroviral therapy has successfully limited HIV-1 disease progression to AIDS, but is consistently compromised by the emergence and transmission of HIV-1 drug resistant strains. As a result, a continued search for novel anti-HIV-1 agents with improved pharmacological profiles has become fundamental. Chrysotherapy has been in use since the early 1920s in treatment of rheumatoid arthritis, and has since been investigated for various ailments including HIV/AIDS. This study evaluated 45 synthetic gold compounds for drug like properties using theoretical and experimental techniques with the aim of generating sufficient data to considerably aid the rational design of new anti-HIV agents. Theoretical techniques applied included the Osiris Property Explorer and the Lipinski’s Rule of Five which assessed drug-likeness and bioavailability respectively. In vitro studies included aqueous solubility assays, cytotoxicity (PM1 cell lines and PBMCs) assays, antiviral assays in PBMCs, direct enzyme (RT and IN) inhibition, and the effect of serum protein binding and biological stability on antiviral efficacy. An overall low druglikeness score and an intermediate bioavailability were predicted by the Osiris Molecular Property Explorer. Low drug-likeness was suggested to be due to a high frequency of foreign fragments in the synthetic gold compounds, while their high molecular weight reduced bioavailability. In general gold compounds exhibited cytotoxicity properties and moderate aqueous solubility in vitro. Overall, the 45 synthetic gold compounds did not show activity against HIV-1 replication in vitro. Seven compounds (AB05-AB11) exhibited direct HIV-1 RT inhibition, and compounds AB39 and AB04 demonstrated moderate direct HIV-1 IN inhibition, but this activity was abrogated in PBMC inhibition assays. Serum binding, compound stability and cytotoxicity were all implicated in the lack of HIV-1 inhibition in PBMCs. To this end, data obtained was sufficient to aid in the future rational design of second generation HIV RT and IN inhibitors with acceptable pharmacological properties.

Mutations in five protein components of the pre-replication complex (pre-RC) have been identified in patients diagnosed with Meier-Gorlin syndrome (MGS), a disorder of global extreme growth failure. ORC1, the most commonly mutated protein, is the largest of six subunits in the origin recognition complex which associates with genomic replication origins and initiates the assembly of the pre-RC during G1 of the cell cycle. Mutations in ORC1, therefore, are expected to perturb cell cycle progression resulting in lengthened cell cycle which leads to a reduction in overall cell number generated during embryogenesis. In this thesis I investigate the developmental and cellular consequences of mutations in ORC1. To address the role of ORC1 during mammalian development I utilised CRISPR/cas9 technology to generate an allelic series of Orc1 mutations in mouse embryonic stem cells (mESCs). Mouse embryos generated by tetraploid complementation assays, using hypomorphic Orc1-mESCs, indicates that impaired Orc1 function in mouse embryogenesis results in developmental delay and a reduction in embryo size. While, no change in proliferation dynamics was seen in Orc1-mESCs in vitro, despite the markedly shortened cell cycle in such cells. Similarly, no significant differences in cell cycle or proliferation rate were detectable in primary patient-derived fibroblasts and lymphoblastoid cell lines. In contrast, Orc1-deficient mouse embryonic fibroblasts had a lengthened cell cycle, as a result of a prolonged G1 phase, implicating the role of the replication licensing checkpoint in somatic cells in lengthening of the cell cycle. In addition, the impact of complete loss of ORC1 was investigated in the developing limb bud. Generation of an Orc1 conditional allele provided a tool to analyse how loss of ORC1 would alter cell cycle in the developing limb. ORC1 is required for embryonic development, and homozygous deletion is embryonic lethal. Mesoderm specific deletions in Orc1 in the developing limb bud using the Prx1-cre transgene resulted in complete loss of forelimb structures, and oligodactyl of the hind limb. Finally, a novel disease gene was identified in patients with MGS. Mutations were identified in 9 patients (7 families) with mutations in CDC45. CDC45 acts downstream of the pre-RC and is required for the formation of the pre-initiation complex, origin activation and fork elongation. As CDC45 acts in the same pathway as ORCs, CDC6 and CDT1, it was hypothesised that hypomorphic mutations in CDC45 might also result in MGS due to impaired DNA replication.

The yeast two-hybrid system was used to examine interactions between the nine proteins involved in baculovirus DNA replication. From the six proteins required for DNA replication, four protein-protein interactions were identified, including an interaction between LEF-1 and LEF-2, LEF-3 and itself, LEF-3 and P143 (Helicase), and an interaction between IE-1 and itself. The replication factors LEF-1 and LEF-2 interacted in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. Using the yeast two-hybrid system, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Deletion analysis of LEF-1 failed to reveal an interaction domain, suggesting that either multiple interaction domains exists or the deletions disrupted secondary structures required for the interaction. All of the deletions which were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction plays a significant role in DNA replication. The baculovirus single-stranded DNA binding protein, LEF-3, interacts with itself in yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. Deletions of LEF-3, which were unable to interact with full length LEF-3, also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to apparent homogeneity and analyzed by analytical ultracentrifugation, native PAGE and MALDI mass spectrometry, identifying the oligomeric structure of LEF-3 as a homotrimer. In addition to interacting with itself, LEF-3 also interacts with P143 in yeast two-hybrid assays, immunoprecipitation experiments, and co-purification from a single-stranded DNA agarose column. The yeast two-hybrid system was used to map the LEF-3 interaction domain to the N-terminal 165 amino acids of LEF-3. Deletion analysis of P143 failed to reveal a delimited interaction domain. C-terminal deletions of LEF-3 containing amino acids 1 to 165 were unable to interact with full length LEF-3, indicating that the interaction of LEF-3 with itself (trimerization) is not required for the interaction between LEF-3 with P143. / Graduation date: 1998