Mechanisms Of Genome Instability

基因组不稳定的机制

• 批准号: 8553734
• 负责人: MICHAEL A RESNICK
• 金额: $ 153万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553734
• 关键词: Acute; Address; Affect; Agreement; Alleles; Aneuploidy; Base Excision Repairs; Biochemical; Bypass; CHEK2 gene; Catalytic Domain; Cells; Chemicals; Chromosomal Instability; Chromosomal Stability; Chromosome Structures; Chromosomes; Chronic; Code; Collaborations; Complex; Cytosine; Cytosine deaminase; DNA Repair; DNA Sequence Rearrangement; DNA biosynthesis; Data; Defect; Development; Diploidy; Disease; Double Strand Break Repair; Environmental Risk Factor; Eukaryota; Event; Evolution; Excision; Excision Repair; Exhibits; Exposure to; Family; Fungal Genome; Gene Dosage; Gene Silencing; Generations; Genes; Genetic; Genetic Nondisjunction; Genetic Recombination; Genetic Risk; Genetic Transcription; Genetic Variation; Genome; Genome Stability; Genomic Instability; Goals; Guanine; Haploidy; Head and Neck Squamous Cell Carcinoma; Health; Hereditary Disease; Homologous Gene; Human; Intervention; Joints; Knowledge; Lead; Lesion; Life; Maintenance; Malignant Neoplasms; Metabolic; Methyl Methanesulfonate; Mismatch Repair; Mitosis; Modeling; Modification; Molecular; Monitor; Multiple Myeloma; Mutagenesis; Mutagens; Mutate; Mutation; Mutation Analysis; Mutation Spectra; Natural Immunity; Nucleotide Excision Repair; Nucleotides; Organism; Pathway interactions; Polymerase; Process; Prostate carcinoma; Proteins; Pulsed-Field Gel Electrophoresis; Recovery; Reporter; Reporter Genes; Resistance; Resolution; Rest; Ribonucleotide Reductase; Ribonucleotide Reductase Inhibitor; Risk; Risk Factors; Role; Saccharomyces cerevisiae; Saccharomycetales; Single base substitution; Single-Stranded DNA; Sister Chromatid; Site; Source; Specificity; Staging; Stress; Sulfites; Sulfur Oxides; System; Temperature; Time; Tissues; Topoisomerase; UV Radiation Exposure; UV induced; Ultraviolet Rays; Uracil; Ursidae Family; Virus; Yeasts; adduct; base; carcinogenesis; cell type; chromosome loss; cohesin; cohesion; density; environmental agent; genome sequencing; hazard; high risk; hydroxyurea; improved; mutant; novel; prevent; recombinational repair; repaired; response; restoration; stem; telomere; transmission process; tumor; ultraviolet damage

DAMAGE-INDUCED LOCALIZED HYPERMUTABILITY (LHM). Mutations are important in evolution as well as many diseases. While mutations are generally considered to accumulate independently, most single base substitutions in coding sequences fail to significantly alter the activity of the corresponding protein. Multiple mutations may be needed to produce dramatic genetic consequences such as gene inactivation or generation of alleles with novel function. 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 yeast grown in the presence of methylmethane sulfonate (MMS). Chronic exposure to MMS caused joint inactivation of the forward mutation reporters URA3 and CAN1 when they were close (separated by 1 kb) but not when they were separated by 85 kb, indicating double mutations occurred primarily via a single localized event. Whole genome sequencing was improved to a level where single base substitutions in 85% of the yeast genome could be detected (collaboration with Dr. Piotr Mieczkowski). Surprisingly, inactivation of URA3 and CAN1 is often accompanied by additional mutations (up to 30) in clusters that span up to 250 kb. The cluster densities were as high as 1/kb. Unlike mutations in the rest of the genome, clusters were predominantly composed of mutations of G:C pairs and contained a strand bias consistent with the mutation spectra of error-prone TLS occurring during restoration of MMS-damaged ssDNA. This base specificity and strand bias indicates DSB associated strand-resection as a major pathway for the LHM in wild type cells. We have also identified a second pathway where mutation clusters occur in ssDNA generated in the mutant cell lacking tof1/timeless-csm3/tipin replication fork protection complex. These smaller clusters (spanning only a few kB) likely stem from broken or uncoupled replication forks. Thus, we identified two pathways of damage-induced mutagenesis in which the combination of localized inability to repair DNA damage along with error-prone translesion synthesis leads to localized severe genetic alteration within a single generation. This scenario could result in rapid diversification and selective advantage in adaptive evolution. It also identifies a possible new source of genetic disease and cancer. Our analysis of mutations found by whole-genome sequencing in several dozens of different tumors have revealed clusters of simultaneous mutations in three types of human cancers, multiple myelomas, prostate carcinomas and in head and neck squamous cells carcinomas. 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. These data indicate that hyper-mutation via multiple simultaneous changes in randomly formed ssDNA is a general phenomenon that may be an important mechanism producing rapid genetic variation in cancers as well as in normal somatic tissues. 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. GENE DOSAGE OF GENOME STABILITY GENES. The sister chromatid cohesion (SCC) complex is involved in chromosome transmission, chromosome structure, maintenance, transcription DNA repair, and cohesin mutations are associated with cancer and developmental defects. We have extended initial findings about cohesin gene dosage to address the role of regulators and the mode of establishment of SCC in genome stability. We explored the cohesin complex per se (Mcd1) and its regulator Wpl1 and found that they prevent misrouting of recombinational DSB repair into break-induced replication (BIR). Haploid and diploid yeast carrying a deletion of WPL1 or a temperature-sensitive mutation mcd1-1 in an essential cohesin subunit have increased BIR and chromosome loss over WT. The mcd1-1 or wpl1 deletion diploids exhibited a dramatic increase (up to 1000-fold) in chromosomal nondisjunction and amplification, resulting in cells with 4 to 5 copies of the reporter chromosome. We propose that the SCC maintenance complex (Wpl1) prevents chromosome instability caused by breakage primarily through limiting BIR, while the core cohesin complex maintains chromosome stability by keeping nondisjunction of unbroken chromosomes as well as BIR at low levels. Using a tetraploid gene dosage model in which only one copy of the yeast RAD53 is functional (simplex), we found that the simplex strain was not sensitive to acute UV radiation or chronic MMS exposure. However, the simplex strain was sensitized to chronic exposure of the ribonucleotide reductase inhibitor hydroxyurea (HU). The importance of this finding is stressed by the fact that the Rad53, the homolog of human Chk2, is a central component of the DNA damage checkpoint system. Surprisingly, reduced RAD53 gene dosage did not affect sensitivity to HU acute exposure, indicating that immediate checkpoint responses and recovery from HU-induced stress were not compromised. We propose that a modest reduction in Rad53 activity can impact the activation of the ribonucleotide reductase catalytic subunit Rnr1 following stress, reducing the ability to generate nucleotide pools sufficient for DNA repair and replication.

损伤诱导的局部超显性（LHM）。突变在进化和许多疾病中很重要。虽然通常认为突变是独立积累的，但编码序列中的大多数单基本取代都无法显着改变相应蛋白的活性。可能需要多个突变来产生巨大的遗传后果，例如基因失活或具有新功能的等位基因的产生。我们发现，瞬时单链DNA（ssDNA）的病变特别威胁到基因组稳定性并导致多个突变的簇。继续我们先前对LHM的研究，我们发现在存在甲基甲烷磺酸盐（MMS）存在下生长的酵母中可能发生突变簇。慢性暴露于MMS导致正向突变记者的关节失活URA3和CAN1接近时（分别为1 kb），但在将它们分离为85 kb时，并非没有，这表明双重突变主要通过单个局部化事件发生。将整个基因组测序提高到可以检测到85％酵母基因组中的单个基本取代的水平（与Piotr Mieczkowski博士的合作）。令人惊讶的是，在跨度为250 kb的簇中，URA3和CAN1的灭活通常伴随着其他突变（最高30）。簇密度高达1/kb。与基因组的其余部分中的突变不同，簇主要由G：C对的突变组成，并包含与在MMS受损的ssDNA恢复过程中发生的易依错误TL的突变光谱一致的链偏差。这种基本特异性和链偏置表明DSB相关的链分离是野生型细胞中LHM的主要途径。 我们还确定了第二个途径，其中突变簇在缺乏TOF1/timeless-CSM3/Tipin复制叉保护配合物的突变细胞中产生的ssDNA中发生。 这些较小的簇（仅跨越少量KB）可能源于破裂或未耦合的复制叉。 因此，我们确定了损伤诱导的诱变的两种途径，其中局部无法修复DNA损伤以及容易发生的跨性别transemion合成的组合会导致单代局部严重的遗传改变。这种情况可能会导致自适应演化的快速多元化和选择性优势。它还确定了可能的新遗传疾病和癌症来源。 我们对数十个不同肿瘤中全基因组测序发现突变的分析揭示了三种类型的人类癌症，多种骨髓瘤，前列腺癌以及头部和颈部鳞状细胞癌中的同时突变簇。与酵母中的发现一致，在重排断点附近经常发现簇。 突变的胞嘧啶或鸟嘌呤的链协调簇高度富集，该基序是由APOBEC家族靶向的ssDNA特异性胞嘧啶脱氨酸酶，涉及对病毒的先天免疫。这些数据表明，通过多个随机形成的ssDNA的多个同时变化超女，是一种普遍现象，它可能是产生癌症以及正常体细胞组织中遗传变异快速遗传变异的重要机制。 为了评估环境药物对染色体ssDNA造成的潜在危害，我们设计了一个在发芽酵母中的ssDNA特异性诱变报告基因。 报告菌株具有CDC13-1温度敏感的突变，使得转移到37oC会导致端粒解膜并随后的5至3切除。 切除导致长ssDNA区域，其中包含3个紧密间隔的报告基因。 我们表征了亚硫酸盐的ssDNA诱变作用，亚硫酸盐是一种经常暴露于人类的一类反应性硫氧化物。我们发现，硫酸盐在DNA中形成了长期寿命的加合性的2-脱氧核心中间体，在修复过程中，在修复过程中，在修复合成聚合酶，最常见的POL ZETA，在修复过程中，在修复合成过程中必须绕过在修复合成过程中对抗切除酶的抗性。 我们的结果表明，硫酸盐诱导的ssDNA病变可能特别有害，因为细胞没有准确修复或绕过此类病变的手段。 此外，该系统还提供了一个机会，以解决受潜在诱变剂挑战时单链DNA与基因组稳定性的相关性。 我们检查了ssDNA的影响，在切除修复过程中可能会作为间隙以及紫外线暴露后的重组可能出现的间隙。使用我们的脉冲场凝胶电泳方法来检测非常缓慢的DNA修复中间体（SMD），并在圆形染色体中对姐妹 - 染色剂重组进行实时监测，我们研究了紫外线损伤后的间隙填充过程，引起了修复途径的重组和协调。在缺乏TLS聚合酶（POL-ETA和POL-ZETA）的突变体中，SMD的量和分辨率的时间增加了，并且在没有TLS的情况下需要进行紫外线修复。因此，紫外线可以在非复制的G2阶段诱导重组，并随着间隙填充过程中的缺陷大大增加。重组维修需要5'至3'EXO1，可提供依赖切除的间隙扩展。此外，拓扑异构酶TOP3促进了紫外线诱导的重组，我们提出，这有助于链条入侵过程，该过程是RAD51和RAD52的上游。总的来说，这些结果表明了一种新型的重组机制，并揭示了ssDNA间隙的复杂且高度协调的修复轮廓。 基因组稳定基因的基因剂量。姊妹染色质被凝聚（SCC）复合物参与染色体传递，染色体结构，维护，转录DNA修复和粘着蛋白突变与癌症和发育缺陷有关。我们扩展了有关粘着素基因剂量的初步发现，以解决调节剂的作用以及SCC在基因组稳定性中的建立方式。我们探索了粘蛋白复合物本身（MCD1）及其调节剂WPL1，发现它们可以防止重组DSB修复误插入破裂引起的复制（BIR）。在必要的粘着素亚基中，携带WPL1或温度敏感的MCD1-1的单倍体和二倍体酵母在WT上的BIR和染色体损失增加。 MCD1-1或WPL1缺失二倍体在染色体非结合和扩增中表现出急剧增加（最高1000倍），从而导致细胞具有4至5份报告基因染色体的副本。 我们建议SCC维护复合物（WPL1）可防止主要是由限制BIR损坏引起的染色体不稳定性，而核心粘连蛋白复合物通过保持无损染色体的非分离以及BIR的非分离来保持染色体稳定性。 使用四倍体基因剂量模型，其中只有一个酵母Rad53的副本是功能性的（单纯形），我们发现单纯形应变对急性UV辐射或慢性MMS暴露不敏感。 然而，单纯菌株对核糖核苷酸还原酶抑制剂羟基脲（HU）的慢性暴露敏感。 这一发现的重要性是由于人类CHK2的同源物Rad53是DNA损伤检查点系统的核心组成部分。 令人惊讶的是，降低的RAD53基因剂量不会影响对HU急性暴露的敏感性，这表明立即检查点响应和从HU诱导的应激中恢复的敏感性不会受到损害。我们认为，在应激之后，Rad53活性的适度降低会影响核糖核苷酸还原酶催化亚基RNR1的激活，从而降低了产生足以用于DNA修复和复制的核苷酸池的能力。