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. 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 chromatin assembly in the suppression of genome instability, 2) Screen more yeast genome instability suppressor genes and 3) the role of human Ku protein in suppression of genome instability 1) The role of yeast chromatin assembly factors in the mantenance of genome stability A number of studies have suggested that spontaneous genome instability can reslut from errors during DNA replication that possibly lead to stalled or broken replication forks. Many pathways seem to act on these errors to promote their correct repair and prevent their conversion into genome instability. Among these pathways are 1) at least three different checkpoints that act during DNA replication S phase, 2) recombination pathways similar to those that promote break-induced replication, 3) a pathway that prevents de novo addition of telomeres to broken DNAs, and 4) possibly mismatch repair that prevents recombination between divergent DNA sequences. The close relationship and coordinated regulation between DNA replication and chromatin assembly are required for normal cell growth. To know whether this coordination is required for the genome stability, yeast strains defective in chromatin assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) were made and their chromosome instability were tested. Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated de novo telomere addition. Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, wherease inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints. Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of de novo telomere addition. Treatment of CAF-I- or RCAF-defective cells with methyl methanesulfonate (MMS) increased the induction of gross chromosomal rearrangemetns (GCRs) compared with that seen for a wild-type strain. These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability. The results accumulated in this study were published in Proceedings of National Academy Science. 2) Screen more yeast genome instability genes. To experimentally expand our knowledge whether there are other pathway(s) that suppress GCRs, we developed a new generally applicable genome wide screening method. In this screen, we identified 8 new genes (CDC50, CSM2, ELG1, ESC1, MMS4, RAD5, RAD18 and TSA1) that encode proteins functioning in the suppression of GCRs. ELG1 was first identified in a screen for mutations that elevate Ty1 cDNA-mediated mobility. Recently, an alternate replication factor complex (RFC) containing ELG1 that is different from RFC1, RAD24 or CTF18 RFCs and its role in DNA replication and checkpoints were reported. ELG1 mainly suppressed de novo telomere addition GCRs. Consistent with this hypothesis, an introduction of mutation in EST2 into the elg1 mutant significantly reduced the GCR rate observed in the elg1 strain indicating that telomerase is required for most elg1 induced GCR. The RAD5 and RAD18 genes encode proteins functioning in post-replication DNA repair. Mutations in either RAD5 or RAD18 increased the GCR rate up to 200 fold. The MMS4 gene was previously identified through a screen for sensitivity to MMS. Subsequent studies found that MMS4 and MUS81 form a heterodimer endonuclease complex that plays a role in processing lesions that block DNA replication, possibly through stalled replication forks during S phase. Similar to the MMS4 mutation, mutation of the MUS81 gene increased the GCR rate almost 200 fold Compared to the ELG1, RAD5, RAD18 and MMS4 genes, there are few studies implicating the CDC50, CSM2, ESC1 and TSA1 genes in DNA metabolism. CDC50 is a cell cycle protein localized mainly in the cytoplasm and involved in protein maturation and transport. MMS and hydroxyurea sensitivities conferred on yeast cells by cdc50 mutation suggest a possible role for CDC50 in DNA metabolism linked to cell cycle regulation. CSM2 functions during meiosis and is required for proper segregation of chromosomes. Inactivation of the CSM2 gene increased the GCR rate 8 fold in the wild type background and 457 fold in the pif1-m2 background. ESC1 encodes a nuclear protein that makes a complex with the SIR4 protein to silence chromatin. Although there was only a 7 fold increase in the GCR rate in the esc1 strain, when the esc1 mutation was combined with a pif1 mutation, a 314 fold increase in the GCR rate was observed. Similarly, combining a tsa1 mutation with the pif1-m2 mutation caused a synergistic increase in the GCR rate. The fact that TSA1 protects cells from free-radical damage suggests that deletion of the TSA1 gene may increase spontaneous DNA damage and as a result increased GCRs. Currently, we are investigating individual genes how they orchestrate to suppress genome instability with different genetic and biochemical methods. 3) The role of human 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 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. Currently, we are investigating the mechanism how Ku protein function to suppress GCRs through telomere length regulation.
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