Genome instability is a characteristic of cancer cells. Different types of genome instability such as the accumulation of mutations, genome rearrangements and aneuploidy have been observed in different genetic disorders including cacners. There is growing evidence supporting the idea that acquisition of a mutator phenotype is required to account for the high rate of accumulating genetic changes in cancer cells. Some of the many examples are cancer susceptibility genes such as ATM, NBS, BLM, BRCA1 and BRCA2 whose protein products have been linked to defects in DNA damage responses and/or DNA repair. In some cases, tumors and even normal blood cells from mutation-bearing individuals show an abnormally high frequency of chromosomal aberrations. While there are many observations of genetic cancer syndromes associated with genome instability, there is little work linking these gene defects to the molecular events that cause genome instability. To understand the mechanisms of genome instability, we have studied 1) The role of Ku protein in suppression of GCR through telomere maintenance, 2) The role of mitotic checkpoint to increase the GCRs and 3) Characterization of MRX (Mre11-Rad50-Xrs2) complex for its role of suppression of GCRs. 1) The role of Ku protein in suppression of GCR through telomere maintenance. Telomeres are the terminal structures of linear chromosomes. Telomeres appear to perform at least two functions; a) they allow for the replication of the ends of chromosomes and b) they stabilize chromosomes by keeping them from recombining with one another. Ku86 plays a key role in nonhomologous end joining (NHEJ) in organisms as evolutionarily disparate as bacteria and humans. In eukaryotic cells, Ku86 has also been implicated in the regulation of telomere length although the effect of Ku86 mutations varies considerably between species. Indeed, telomeres either shorten significantly, shorten slightly, remain unchanged, or lengthen significantly in budding yeast, fission yeast, chicken cells or plants, respectively, that are null for Ku86 expression. Thus, it has been unclear which model system is most relevant for humans. We found that the functional inactivation of even a single allele of Ku86 in human somatic cells results in profound telomere loss, which is accompanied by an increase in chromosomal fusions, translocations and genomic instability. Together, these experiments demonstrate that Ku86, separate from its role in nonhomologous end joining, performs that additional function in human somatic cells of suppressing genomic instability through the regulation of telomere length. Furthermore, we investigated the mechanism how Ku proteins suppress genome instability by using yeast GCR assay system. We found that the overexpression of yeast Ku70-Ku86 heterodimer suppressed the GCR formation either spontaneously generated or induced by treatments with different DNA damaging agents, which are sometimes used for radio- and chemotherapies for cancer. The suppression of GCR formation by the yKu70-yKu86 overexpression was disappeared only when the DNA damage checkpoint is despaired suggesting that the GCR suppression by Ku protein is its interaction through the DNA damage checkpoint not through its role in NHEJ. The Ku overexpression caused cell growth delay, which is dependent on intact Okazaki fragment maturation proteins. Furthermore, the inactivation of telomerase inhibitor, Pif1 along with Ku overexpression arrested cell cycle at S phase in DNA damage checkpoint dependent fashion. 2) The role of mitotic checkpoint to increase the GCRs. Checkpoints are surveillance mechanisms designed to ensure correct transmission of genetic information during cell division. There are a number of checkpoints that respond to DNA damage and as well as aberrant DNA structures that occur when DNA replication is blocked. The DNA damage checkpoint arrests the cell cycle eithher in G1 or G2 in response to DNA damage and also results in the slowing of DNA replication when DNA damage occurs during S phase; this latter checkpoint response is sometimes called the intra-S checkpoint. The DNA replication checkpoint arrests cell cycle progression and suppresses the firing of late replication origins in response to blocked DNA replication. The mitotic checkpoints respond to the failure of spindle assembly and arrests the cell cycle at M phase. Genetic defects in various DNA damage and S-phase checkpoints have been demonstrated to result in differing degrees of increased spontaneous GCR rates, increased chromosome loss and increased recombination. However, compared to the significant increases in GCR rates caused by defects in S phase checkpoints including the replication checkpoint and intra-S checkpoints, defects in the mitotic checkpoint did not appear to increase GCR rates. The mitotic checkpoint, also known as the spindle checkpoint, ensures proper chromosome segregation by arresting the cell cycle at mitosis by responding to improper or incomplete spindle assembly. In S. cerevisiae the genes that function in the mitotic checkpoint include MAD1, 2, 3, BUB1, 3 and MPS1 and these in part function through the anaphase-promoting complex (APC). Bub2 functions in the MEN by inhibiting the degradation of mitotic cyclins and other regulators of the exit from mitosis. Mitotic exit is achieved by inactivation of Tem1 by conversion of bound GTP to GDP by the Bub2 and Bfa1 GTPase activating proteins. Mutations in genes encoding mitotic checkpoint and MEN proteins lead to increased missegregation of chromosomes even in the absence of spindle damages and failure of mitotic cell cycle arrest in response to spindle depolymerizing drugs such as nocodazole or benomyl. We found that defects in the mitotic checkpoint and the mitotic exit network (MEN) often result in the suppression of GCRs in strains containing defects that increase the GCR rate. These data strongly suggest that functional mitotic checkpoints can play an important role in the formation of genome rearrangements. 3) Characterization of MRX (Mre11-Rad50-Xrs2) complex for its role of suppression of GCRs. Mutation in any of genes in the MRX complex (MRE11, RAD50 or XRS2) causes sensitivity to alkylating agents and ionizing radiation, defects in mitotic and meiotic recombination and NHEJ and also results in a decrease in telomere length. The fact that the mammalian MRX complex equivalent forms foci at the site of DNA damage suggests that the MRX complex functions in cell cycle checkpoints and DNA repair in mammalian cells. Furthermore, mutations of genes encoding these subunits have been identified in many cancer prone syndromes including Ataxia Telangiectasia Like Disorder (ATLD) and Nijmegen Breakage Syndrome (NBS). Null mutations in any of the MRX genes increased the GCR rate up to 1000 fold. However, because of the multiple functions and biochemical activities of the MRX complex, it is unclear which functions and what biochemical activities of the MRX complex are important for suppression of GCRs. To understand which function of MRX complex is the main function of GCR suppression, we generated different types of point mutations that specifically inactivate different MRX functions. We found that at least three different activities of the MRX complex are important for suppression of GCRs. The nuclease activity of Mre11, an activity related to MRX complex formation and the telomere maintenance function of the MRX complex are important for the suppression of GCRs. An activity related to MRX complex formation is especially important for the suppression of translocation type of GCRs.
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