DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. In this work, I reported the completion of a transposon mutagenesis screen in budding yeast and the identification of five novel genes (RRD1, CIK1, CTF18, RTS1, and IRC6) critical for telomere healing. The characterization of Rrd1 led to the surprising finding that Rrd1 facilitates telomere healing at DSBs with little or no TG-rich sequences but not at DSBs with long tracts of telomeric sequences. Pph3, a PP4 phosphatase, acts in conjunction with Rrd1 to promote telomere healing. Conversely, Mec1, the ATR ortholog, phosphorylates Cdc13 on its S306 residue to suppress its accumulation at DSBs. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DSBs. Next, I found that Cik1 and its kinesin partner Kar3 are both important for telomere healing. Importantly, Kar3 contributes to telomere healing through its motor function. In contrast to Rrd1, Kar3 contributes to telomere healing regardless of telomeric sequence lengths adjacent to the break. Finally, Cik1 and Kar3 have a general role in DNA repair and physically associate with DSBs, which is dependent on the process of anchoring DSBs to nuclear periphery. In conclusion, I identified a mechanism by which the ATR family of kinases enforces genome integrity, a phosphoregulatory loop that underscores the contribution of Cdc13 to the fate of DNA ends, and a kinesin complex critical for the spatial organization of DNA repair.

DNA lesions arise constantly in cells and are repaired by a variety of DNA repair pathways. Polynucleotide kinase 3’-phosphatase (PNKP) aids repair by phosphorylating 5’-hydroxyl DNA termini and dephosphorylating 3’-phosphate DNA termini for the completion of repair by DNA ligases. This activity is critical in vivo because DNA breaks do not usually possess ligatable termini. PNKP knockdown sensitizes cells to several DNA damaging agents, including the topoisomerase I (TOP1) inhibitor camptothecin - analogs of which are being developed into chemotherapeutic drugs - because the resolution of stalled TOP1-DNA complexes requires processing by PNKP. We hypothesize that small molecule inhibitors of PNKP could bolster the effects of radio- and chemotherapies on cancer cells. I have identified eight compounds that effectively inhibit human PNKP and, with reduced potency, T4 PNK in vitro. These compounds act by reversibly inhibiting the substrate-enzyme interaction but they do not appear to sensitize U2OS cells to camptothecin.

DNA lesions arise constantly in cells and are repaired by a variety of DNA repair pathways. Polynucleotide kinase 3’-phosphatase (PNKP) aids repair by phosphorylating 5’-hydroxyl DNA termini and dephosphorylating 3’-phosphate DNA termini for the completion of repair by DNA ligases. This activity is critical in vivo because DNA breaks do not usually possess ligatable termini. PNKP knockdown sensitizes cells to several DNA damaging agents, including the topoisomerase I (TOP1) inhibitor camptothecin - analogs of which are being developed into chemotherapeutic drugs - because the resolution of stalled TOP1-DNA complexes requires processing by PNKP. We hypothesize that small molecule inhibitors of PNKP could bolster the effects of radio- and chemotherapies on cancer cells. I have identified eight compounds that effectively inhibit human PNKP and, with reduced potency, T4 PNK in vitro. These compounds act by reversibly inhibiting the substrate-enzyme interaction but they do not appear to sensitize U2OS cells to camptothecin.

DNA is susceptible to both exogenous and endogenous damaging agents. Damage is constantly reversed by a wide range of DNA repair pathways. Lesions which escape such repair may cause nucleotide mis-pairing and stalled replication, resulting in mutagenesis and cell death, respectively if left unresolved. Stalled replication is particularly dangerous because replication fork collapse can lead to double-strand breaks (DSBs) and chromosome rearrangement, a hallmark of cancer. DNA damage tolerance (DDT) is defined as a mechanism that allows DNA synthesis to occur in the presence of replication-blocking lesions.<p> DDT, also known as post-replication repair (PRR) in yeast, has been well characterized in the lower eukaryotic model Saccharomyces cerevisiae to consist of error-free and error-prone (mutagenic) pathways. Mono-ubiquitination of proliferating cell nuclear antigen (PCNA) by the Rad6-Rad18 complex promotes mutagenesis by recruiting low fidelity translesion synthesis (TLS) polymerases, while continual Lys63-linked poly-ubiquitination of PCNA by the Mms2-Ubc13-Rad5 complex promotes error-free lesion bypass. Since most of the genes involved in DNA metabolism are conserved within eukaryotes, from yeast to human, I tested the hypothesis that mammalian cells also possess two-pathway DDT in response to DNA damage. Namely, the error-free pathway is dependent on the Ubc13-Mms2 complex, while the error-prone pathway utilizes the TLS polymerases, such as Rev3.<p> By utilizing cultured mammalain cells and producing antibodies against human Ubc13, Mms2 and Rev3, I was able to show that all three proteins associate with PCNA in S-phase cells, and that this association is enhanced following DNA damage. Ubc13-Mms2 association with PCNA was enhanced in response to DSBs. Furthermore, suppression of Ubc13 or Mms2 using interfering RNA technology resulted in increased spontaneous DSBs. In response to UV exposure, Rev3 co-localized with PCNA and two other TLS polymerases, Rev1 and Pol-Ø, at the damage site. UV-induced Rev3 nuclear focus formation was dependent on Rev1 but independent of Pol-£b. Surprisingly, over-expression of Pol-£b was sufficient to induce spontaneous Rev3 nuclear foci. It was further demonstrated that Rev1 and Pol-Ø were independently recruited to the damage site and did not require Rev3. These observations support and extend the polymerase switch model which regulates the activity of the replicative and TLS polymerases. Finally, simultaneous suppression of Rev3 along with Ubc13 or Mms2 resulted in a synergistic sensitivity to UV, whereas simultaneous suppression of Ubc13 and Pol-Ø resulted in an additive effect. These results are consistent with those in yeast cells, implying a comparable mammalian two-pathway DDT model.<p> Additional interesting observations were made. Firstly, Ubc13 interacts with Uev1A, a close homolog of Mms2, which is involved in the NF-£eB signaling pathway independent of DNA damage. Secondly, Rev3 appears to be excluded from the nucleus in a fraction of low passage normal non-S-phase cells, whereas in tumor derived cell lines, Rev3 is consistently enriched in the nucleus independent of cell cycle stage. Finally, Rev3 is elevated during mitosis and associates with condensed chromosomes, suggesting a possible novel role in mitosis. Consistent with this notion, chronic ablation of Rev3 resulted in cell death with inappropriate chromosome segregations. The above preliminary observations require further investigation.

DNA damage represents a major challenge to the faithful replication and transmission of genetic information from one generation to the next. Cells utilize a highly integrated network of pathways to detect and accurately repair DNA damage. Mutations arise when DNA damage persists undetected, unrepaired, or repaired improperly. Mutations are a driving force of carcinogenesis and therefore many of the DNA damage surveillance and repair mechanisms guard against the transformation of normal cells into cancer cells. Central to the detection and repair of DNA damage is the relocalization of DNA damage surveillance proteins to DNA damage where they assemble into subnuclear foci and are capable to producing a signal that the cell interprets to induce cellular modifications such as cycle arrest and DNA repair which are important DNA damage tolerance. In this work, I describe my quest to understand the mechanisms underlying the assembly, maintenance, and disassembly of these DNA damage-induced foci and how they affect DNA damage signaling in Saccharomyces cerevisiae. First, I describe phenotypic characterization of a novel mutation that impairs assembly of the 9-1-1 checkpoint clamp complex into foci. Second, I describe my work to further understand the roles of the histone phosphatase Pph3 and phosphorylated histone H2A in modulating DNA damage signaling. Third, I include my work to uncover the possible mechanism by which the helicase Srs2 works to enable termination of DNA damage signaling. In summary, this thesis documents my efforts to understand the cellular and molecular nature of DNA damage signaling and how signaling is turned off in coordination with DNA damage repair.

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. In this work, I reported the completion of a transposon mutagenesis screen in budding yeast and the identification of five novel genes (RRD1, CIK1, CTF18, RTS1, and IRC6) critical for telomere healing. The characterization of Rrd1 led to the surprising finding that Rrd1 facilitates telomere healing at DSBs with little or no TG-rich sequences but not at DSBs with long tracts of telomeric sequences. Pph3, a PP4 phosphatase, acts in conjunction with Rrd1 to promote telomere healing. Conversely, Mec1, the ATR ortholog, phosphorylates Cdc13 on its S306 residue to suppress its accumulation at DSBs. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DSBs. Next, I found that Cik1 and its kinesin partner Kar3 are both important for telomere healing. Importantly, Kar3 contributes to telomere healing through its motor function. In contrast to Rrd1, Kar3 contributes to telomere healing regardless of telomeric sequence lengths adjacent to the break. Finally, Cik1 and Kar3 have a general role in DNA repair and physically associate with DSBs, which is dependent on the process of anchoring DSBs to nuclear periphery. In conclusion, I identified a mechanism by which the ATR family of kinases enforces genome integrity, a phosphoregulatory loop that underscores the contribution of Cdc13 to the fate of DNA ends, and a kinesin complex critical for the spatial organization of DNA repair.

DNA is susceptible to both exogenous and endogenous damaging agents. Damage is constantly reversed by a wide range of DNA repair pathways. Lesions which escape such repair may cause nucleotide mis-pairing and stalled replication, resulting in mutagenesis and cell death, respectively if left unresolved. Stalled replication is particularly dangerous because replication fork collapse can lead to double-strand breaks (DSBs) and chromosome rearrangement, a hallmark of cancer. DNA damage tolerance (DDT) is defined as a mechanism that allows DNA synthesis to occur in the presence of replication-blocking lesions.<p> DDT, also known as post-replication repair (PRR) in yeast, has been well characterized in the lower eukaryotic model Saccharomyces cerevisiae to consist of error-free and error-prone (mutagenic) pathways. Mono-ubiquitination of proliferating cell nuclear antigen (PCNA) by the Rad6-Rad18 complex promotes mutagenesis by recruiting low fidelity translesion synthesis (TLS) polymerases, while continual Lys63-linked poly-ubiquitination of PCNA by the Mms2-Ubc13-Rad5 complex promotes error-free lesion bypass. Since most of the genes involved in DNA metabolism are conserved within eukaryotes, from yeast to human, I tested the hypothesis that mammalian cells also possess two-pathway DDT in response to DNA damage. Namely, the error-free pathway is dependent on the Ubc13-Mms2 complex, while the error-prone pathway utilizes the TLS polymerases, such as Rev3.<p> By utilizing cultured mammalain cells and producing antibodies against human Ubc13, Mms2 and Rev3, I was able to show that all three proteins associate with PCNA in S-phase cells, and that this association is enhanced following DNA damage. Ubc13-Mms2 association with PCNA was enhanced in response to DSBs. Furthermore, suppression of Ubc13 or Mms2 using interfering RNA technology resulted in increased spontaneous DSBs. In response to UV exposure, Rev3 co-localized with PCNA and two other TLS polymerases, Rev1 and Pol-Ø, at the damage site. UV-induced Rev3 nuclear focus formation was dependent on Rev1 but independent of Pol-£b. Surprisingly, over-expression of Pol-£b was sufficient to induce spontaneous Rev3 nuclear foci. It was further demonstrated that Rev1 and Pol-Ø were independently recruited to the damage site and did not require Rev3. These observations support and extend the polymerase switch model which regulates the activity of the replicative and TLS polymerases. Finally, simultaneous suppression of Rev3 along with Ubc13 or Mms2 resulted in a synergistic sensitivity to UV, whereas simultaneous suppression of Ubc13 and Pol-Ø resulted in an additive effect. These results are consistent with those in yeast cells, implying a comparable mammalian two-pathway DDT model.<p> Additional interesting observations were made. Firstly, Ubc13 interacts with Uev1A, a close homolog of Mms2, which is involved in the NF-£eB signaling pathway independent of DNA damage. Secondly, Rev3 appears to be excluded from the nucleus in a fraction of low passage normal non-S-phase cells, whereas in tumor derived cell lines, Rev3 is consistently enriched in the nucleus independent of cell cycle stage. Finally, Rev3 is elevated during mitosis and associates with condensed chromosomes, suggesting a possible novel role in mitosis. Consistent with this notion, chronic ablation of Rev3 resulted in cell death with inappropriate chromosome segregations. The above preliminary observations require further investigation.

<p>A mitotically dividing cell regularly experiences DNA damage including double-stranded DNA breaks (DSBs). Homologous mitotic recombination is an important mechanism for the repair of DSBs, but inappropriate repair of DNA breaks can lead to genome instability. Despite more than 70 years of research, the mechanism of mitotic recombination is still not understood. By genetic and physical studies in the yeast Saccharomyces cerevisiae, I investigated the mechanism of reciprocal mitotic crossovers. Since spontaneous mitotic recombination events are very infrequent, I used a diploid strain that allowed for selection of cells that had the recombinant chromosomes expected for a reciprocal crossover (RCO). The diploid was also heterozygous for many single-nucleotide polymorphisms, allowing the accurate mapping of the recombination events.</p> <p>I mapped spontaneous crossovers to a resolution of about 4 kb in a 120 kb region of chromosome V. This analysis is the first large-scale mapping of mitotic events performed in any organism. One region of elevated recombination was detected (a "hotspot") and the region near the centromere of chromosome V had low levels of recombination ("coldspot"). This analysis also demonstrated the crossovers were often associated with the non-reciprocal transfer of information between homologous chromosomes; such events are termed "gene conversions" and have been characterized in detail in the products of meiotic recombination. The amount of DNA transferred during mitotic gene conversion events was much greater than that observed for meiotic conversions, 12 kb and 2 kb, respectively. In addition, about 40% of the conversion events had patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.</p> <p>To confirm this unexpected conclusion, I examined the crossovers and gene conversion events induced by gamma irradiation in G1- and G2-arrested diploid yeast cells. The gene conversion patterns of G1-irradiated cells (but not G2-irradiated cells) mimic the conversion events associated with spontaneous reciprocal crossovers (RCOs), confirming my hypothesis that many spontaneous crossovers are initiated by a DSB on an unreplicated chromosome. In conclusion, my results have resulted in a new understanding of the properties of mitotic recombination within the context of cell cycle.</p> / Dissertation

In recent decade, lung cancers had the highest incidence and mortality rate among all cancers in Taiwan. Among lung cancers, adenocarcinoma was the most frequent type. The chemotherapy was still the main choice in treating lung cancer by the mechanism of destroying DNA, but the response rate kept low. The function of DNA repair makes cancer cells resistant to chemotherapy. Therefore, this study focused on the effect of cancer cell growth by silencing Mre11. The first part of this study was to make a tissue microarray consisting of adenocarcinoma from 57 patients. Immunohistochemistry staining for Mre11 was done. The correlation of Mre11 expression and clinical variables with survival was analyzed. The second part was tried to knockdown Mre11 in A549 cell by shRNA. Another A549 cell line containing empty vector was selected as control group. These cell lines were then ready for XTT method, soft agar colony formation assay, flow cytometry and nude mice assay. In the clinical data, the absence of lymph node and distant site metastasis were good prognosis factor for longer survival. Although the high expression on Mre11 had longer survival, this variable was not a true independent factor. On XTT method and soft agar colony formation assay, the A549 cells with Mre11 knockdown had a slower proliferation and fewer colony numbers, respectively. The cell cycle demonstrated an elevated G0/G1 and S phase and depressed G2/M phase in A549 cells with Mre11 knockdown. The tumor arising from A549 cells with Mre11 knockdown in the nude mice also had a smaller size. Based on the above study, inhibition of Mre11 may result in a reduction of tumor growth and provide another choice to treat lung cancer.

Thesis (Ph. D.)--University of California, Riverside, 2009. / Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed March 16, 2010). Includes bibliographical references. Also issued in print.