Mechanisms Of Genome Instability

损伤诱导的局部超变性（lhm）。突变在进化和许多疾病中都很重要。我们发现瞬时单链DNA （ssDNA）的损伤尤其威胁到基因组的稳定性，并导致多重突变的集群。继续我们之前对LHM的研究，我们发现突变簇可以发生在多种类型的人类癌症中。与酵母的发现一致，团簇经常在重排断点附近发现。链协调的突变胞嘧啶或鸟嘌呤簇高度富集了一个基序，该基序被参与病毒先天免疫的ssdna特异性胞嘧啶脱氨酶APOBEC家族靶向。通过与布罗德研究所的合作。Gordenin和Roberts参与了对来自癌症基因组图谱（TCGA）的3083对肿瘤-正常对的外显子组序列的突变谱分析。结果是发现了癌症类型中突变频率和频谱的广泛变化，揭示了突变过程和疾病病因。重要的是，在几种癌症类型中发现了一个与APOBEC相似的TC突变特征。在这一观察结果的指导下，我们开发了分析方法来评估来自多个全基因组和外显子组突变数据集（如TCGA）的单个样本中APOBEC突变模式的强度。我们对多种癌症样本中复杂突变谱的统计探索方法涉及围绕诊断突变模式的单一假设的制定，该假设将基于先前实验和数据分析的信息。通过在突变核苷酸周围的+/- 20个核苷酸中存在APOBEC突变基序（TCW或WGA）来计算APOBEC特征突变的富集。我们只利用突变周围的直接环境，因为APOBEC酶被认为扫描有限区域的ssDNA，以在首选序列环境中脱氨C。这种方法不排除基因组的任何给定区域，而是利用突变发生的区域，然后确定突变是否为APOBEC特征富集。APOBEC突变模式在几种癌症类型的许多样本中都很突出，甚至普遍存在，而在其他癌症类型中几乎无法检测到。此外，APOBEC突变的出现与APOBEC mRNA水平相关，并延伸到多种标准认为是癌症驱动因素的基因子集。

DAMAGE-INDUCED LOCALIZED HYPERMUTABILITY (LHM). Mutations are important in evolution as well as many diseases. We found that lesions in transient single-strand DNA (ssDNA) are especially threatening to genome stability and lead to clusters of multiple mutations. Continuing our previous studies of LHM we found that mutation clusters can occur in multiple types of human cancers. In agreement with findings in yeast, clusters were often found in the vicinity of rearrangement breakpoints. Strand-coordinated clusters of mutated cytosines or guanines were highly enriched with a motif targeted by APOBEC family of ssDNA-specific cytosine-deaminases involved in the innate immunity against viruses. In conjunction with the Broad Institute, Drs. Gordenin and Roberts participated in the analysis of mutation spectra in exome sequences from 3,083 tumor-normal pairs, primarily from The Cancer Genome Atlas (TCGA). The outcome was a discovery of wide variation in mutation frequency and spectrum within cancer types, shedding light on mutational processes and disease etiology. Importantly, a TC mutation signature was discovered that was reminiscent of APOBEC in several cancer types. Guided by this observation we developed analytical approaches for evaluating the strength of an APOBEC mutation pattern in the individual samples from multiple whole-genome and exome mutation datasets such as TCGA. Our approach to the statistical exploration of complex mutation spectra in multiple cancer samples involved formulation of a single hypothesis surrounding a diagnostic mutation pattern that would be based on information from prior experiments and data analyses. Enrichment for APOBEC signature mutations was calculated over the presence of the APOBEC mutation motif (TCW or WGA) in the +/- 20 nucleotides surrounding mutated nucleotides. We utilized only the immediate context surrounding mutations because APOBEC enzymes are thought to scan a limited area of ssDNA to deaminate C in a preferred sequence context. This approach does not exclude any given region of the genome, but rather utilizes regions where mutagenesis has occurred and then determines if the mutagenesis is enriched for the APOBEC signature. The APOBEC mutation pattern is prominent and even prevailing in many samples among several cancer types, as opposed to other cancer types where it is hardly detectable. Additionally, the appearance of APOBEC mutations correlates with APOBEC mRNA levels and extends into a subset of genes considered by multiple criteria to be cancer drivers. In order to assess the potential hazard posed by environmental agents to chromosomal ssDNA, we devised a ssDNA-specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature sensitive mutation, such that shifting to 37oC results in telomere uncapping and ensuing 5 to 3 resection. The resection results in long ssDNA regions containing 3 closely-spaced reporter genes. We characterized the ssDNA mutagenic action of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently. We found that sulfites form a long-lived adducted 2-deoxyuracil intermediate in DNA that is resistant to excision by uracil-DNA N-glycosylase and must be bypassed during repair synthesis by a translesion synthesis polymerase, most frequently Pol zeta, during repair synthesis. Our results suggest that sulfite-induced lesions in ssDNA can be particularly deleterious, since cells do not possess the means to repair or bypass such lesions accurately. In addition, this system provides an opportunity to address the relevance of single-strand DNA to genome stability when challenged by potential mutagens. We examined the impact of ssDNA that can arise as gaps during excision repair and possible associations with recombination following UV-exposure. Using our pulsed-field gel electrophoresis approaches for detecting very slow-moving DNA repair intermediates (SMD) and real-time monitoring of sister-chromatid recombination in a circular chromosome, we studied the gap filling process after UV damage, induced recombination and coordination of repair pathways. The amount of SMD and the time required for resolution was increased in mutants lacking TLS polymerases (Pol-eta and Pol-zeta) and recombination was required for UV repair in the absence of TLS. Thus, UV can induce recombination in the nonreplicating G2 stage and is dramatically increased with defects in gap filling process. The 5' to 3' Exo1, which provides excision dependent gap extension, is required for recombination repair. Moreover, the UV-induced recombination was facilitated by the topoisomerase Top3, which we propose assists the strand invasion process that is upstream of Rad51 and Rad52. Collectively, these results suggest a novel mechanism of recombination and reveal a complex and highly-coordinated repair profile of the ssDNA gap. EFFECTS OF COHESIN DEFECTS ON GENOME STABILITY. Gain or loss of chromosomes resulting in aneuploidy or loss-of heterozygosity (LOH) is an important factor in cancer and adaptive evolution. Although chromosome gain is often found in tumors and fungi and is a frequent event in all kingdoms, its genetic control has hardly been studied. We measured the rates of chromosome gain in WT yeast and sister chromatid cohesion (SCC) compromised strains. Even mild deactivation of the cohesion complex caused a high rate of chromosome gain. On top of defects in SCC, yeast cell type had a significant contribution to chromosome gain, with the greatest rates observed for homozygous mating type diploids, then heterozygous mating type and smallest in haploids under all SCC defective strains. We suggest that while chromosome gain due to SCC malfunction can have negative effects through gene imbalance it could also facilitate opportunities for evolution and adaptive changes. In multicellular organisms, both factors could lead to somatic diseases including cancer. The sister chromatid cohesion process (SCC) mediated via the cohesin complex tethers the newly replicated sister chromatids until mitosis and thus suppresses LOH through chromosome loss. SCC is also important to channel sister chromatid recombinational repair to sisters thereby preventing allelic recombination. Until our work, it was unknown how different mutations in the SCC pathway prevent different modes of LOH. We found that the cohesin mutation mcd1-1 and other mutations in SCC differentially affected various types of LOH of a centromeric marker. The effects of mutations were greatest for whole chromosome loss. We suggest that SCC malfunction could lead to extensive LOH and thus facilitate opportunities for evolution and carcinogenesis through homozygosis of many recessive mutations.