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In budding yeast, many of the genes that encode enzymes required for DNA precursor synthesis (MCB genes) are expressed under cell cycle control in late G1/S. The relationship between MCB gene expression, dNTP synthesis and DNA synthesis was investigated by using �� factor-synchronized Saccharomyces cerevisiae. The levels of all four dNTPs increased several-fold when cells crossed the G1/S boundary. An even larger increase in the dNTP pools occurred at G1/S when replication initiation was blocked by incubating synchronized dbf4 mutants at the nonpermissive temperature. Thus, dNTP accumulation at G1/S was not dependent on replication initiation. Similarly, MCB gene induction at G1/S was also independent of replication initiation. The accumulation of dNTPs at G1/S was dependent on Swi6, a protein known to be required for normal MCB gene regulation during the cell cycle. Treatment with hydroxyurea, an inhibitor of ribonucleotide reductase, blocked DNA synthesis and prevented the increase in dNTP levels that normally occurred at G1/S, however, it did not exhaust the basal levels of any of the four dNTPs. The mechanism responsible for replication arrest despite the persistence of dNTPs was not dependent on the checkpoint protein Rad53, as rad53 mutants also failed to exhaust basal dNTPs when incubated in HU. The inhibitory effect of HU on DNA synthesis was bypassed when dbf4 cells were allowed to pre-accumulate dNTPs at 37��C before being released to the permissive temperature in the presence of HU. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, as dbf4 cells incubated in HU at 25��C were able to initiate replication when cells were switched to the nonpermissive temperature and HU was removed. The results indicate that DNA chain elongation in yeast requires a critical dNTP threshold, below which replication forks are completely arrested. Cells lacking a functional thioredoxin system were deficient in dNTP synthesis. The rate of accumulation was significantly lower in ��trr1 mutants lacking thioredoxin reductase, and dNTP accumulation at G1/S did not occur at all in ��trxl ��trx2 double mutants lacking thioredoxin. The results suggest that thioredoxin serves as the electron donor for ribonucleotide reductase during DNA precursor synthesis in yeast. / Graduation date: 2003

In response to DNA damage, the ATM and ATR kinases proliferate a signal that is transduced, either directly or via Chk2 and Chk1, to effector proteins, forming the DNA damage response (DDR). The effector proteins delay cell cycle progression, through checkpoints, and activate specific DNA repair mechanisms essential for preserving genome integrity and preventing cancer formation. Bloom's syndrome (BS) patients, which lack the BLM protein show genome instability and have a predisposition to cancer. BLM is phosphorylated by the DDR kinases ATM, ATR and Chk1. These phosphorylation events are essential for BLM to maintain replication fork integrity, preserve the S phase checkpoint and activate BLM to interact with other DDR proteins. In this study I have shown that BLM, isolated from mitotic cells, is phosphorylated on amino acid residue serine 26 (S26). BS cells lacking native BLM, but expressing a variant of BLM protein that cannot be phosphorylated at S26, fail to fully activate the G2/M checkpoint following UV irradiation or treatment with inhibitors of DNA topoisomerase H. Consequently, these cells are more sensitive to killing by these agents than are BS cells expressing wildtype BLM. The Chk1 and Aurora B kinases are able to phosphorylate BLM on S26 in vitro. Moreover, loss of Aurora B kinase activity leads to reduction of S26 phosphorylation in mitotic cells. Cells treated with inhibitors of Aurora B fail to fully active the G2/M checkpoint after UV DNA damage. Taken together, these data suggest, that Aurora B kinase phosphorylates BLM on S26 and that this is required to fully activate the G2/M checkpoint.

In order to maintain genetic stability in eukaryotes, tight regulation of the relative sizes of deoxyribonucleoside triphosphate (dNTP) levels inside the cell is essential for optimal fidelity of DNA replication. Ribonucleotide reductase (RNR) is the enzyme responsible for proportional production of DNA precursors. Studies on regulation of this enzyme, the focus of this thesis, are important because mutations affecting RNR control mechanisms result in dNTP pool imbalance, thus promoting mutagenesis. By using mouse RNR as a model for mammalian forms of the enzyme, three major factors--allosteric effectors, rNDP substrate concentrations, and hypoxic conditions--that influence the substrate specificity of RNR have been investigated. Allosteric regulation has been studied by the four-substrate assay, which permits simultaneous monitoring of the four reactions catalyzed by this enzyme in one reaction mixture. Individual dNTPs affect the four activities differentially in a concentration-dependent manner with discrete effects of dTTP and dGTP on reduction of ADP and GDP, respectively. Ribonucleoside diphosphate (rNDP) substrate concentrations are equally important, as their variations lead to different product ratios. Results from nucleotide binding assays indicate that rNDPs directly influence binding of dNTP effectors at the specificity site, one of the two classes of allosteric sites, whereas ADP has an indirect effect, displacing other substrates at the catalytic site and consequently removing effects of those substrates upon dNTP binding. Hence, this is the first evidence of a two-way communication between the catalytic site and the specificity site. Oxygen limitation also plays an important role in controlling the enzyme specificity. Reactivation of the enzyme at different oxygen tensions, after treatment of the enzyme with hydroxyurea (HU) followed by removal of HU, reveals a distinct sensitivity of GDP reductase to low 0��� levels. Although the basis for specific inhibition of GDP reduction remains to be determined, some possibilities have been ruled out. This research proves that in addition to allosteric regulation by nucleoside triphosphates, mouse RNR is also controlled by other factors. Since these components can simultaneously exert their effects upon enzyme specificity, complex regulatory patterns of RNR to provide a proportional supply of the DNA building blocks in vivo are suggested. / Graduation date: 2003

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Indiana University-Purdue University Indianapolis (IUPUI) / Targeting uncontrolled cell proliferation and resistance to DNA damaging chemotherapeutics using small molecule inhibitors of proteins involved in these pathways has significant potential in cancer treatment. Several proteins involved in genomic maintenance and stability have been implicated both in the development of cancer and the response to chemotherapeutic treatment. Replication Protein A, RPA, the eukaryotic single-strand DNA binding protein, is essential for genomic maintenance and stability via roles in both DNA replication and repair. Xeroderma Pigmentosum Group A, XPA, is required for nucleotide excision repair, the main pathway cells employ to repair bulky DNA adducts. Both of these proteins have been implicated in tumor progression and chemotherapeutic response. We have identified a novel small molecule that inhibits the in vitro and cellular ssDNA binding activity of RPA, prevents cell cycle progression, induces cytotoxicity and increases the efficacy of chemotherapeutic DNA damaging agents. These results provide new insight into the mechanism of RPA-ssDNA interactions in chromosome maintenance and stability. We have also identified small molecules that prevent the XPA-DNA interaction, which are being investigated for cellular and tumor activity. These results demonstrate the first molecularly targeted eukaryotic DNA binding inhibitors and reveal the utility of targeting a protein-DNA interaction as a therapeutic strategy for cancer treatment.

Indiana University-Purdue University Indianapolis (IUPUI) / Break-induced replication (BIR) is a mechanism to repair double-strand breaks (DSBs) that possess only a single end that can find homology in the genome. This situation can result from the collapse of replication forks or telomere erosion. BIR frequently produces various genetic instabilities including mutations, loss of heterozygosity, deletions, duplications, and template switching that can result in copy-number variations (CNVs). An important type of genomic rearrangement specifically linked to BIR is half crossovers (HCs), which result from fusions between parts of recombining chromosomes. Because HC formation produces a fused molecule as well as a broken chromosome fragment, these events could be highly destabilizing. Here I demonstrate that HC formation results from the interruption of BIR caused by a defective replisome or premature onset of mitosis. Additionally, I document the existence of half crossover instability cascades (HCC) that resemble cycles of non-reciprocal translocations (NRTs) previously described in human tumors. I postulate that HCs represent a potent source of genetic destabilization with significant consequences that mimic those observed in human diseases, including cancer.