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.

损伤诱导的局部超变性（lhm）。突变在进化和许多疾病中都很重要。虽然突变通常被认为是独立积累的，但大多数编码序列中的单碱基替换不能显著改变相应蛋白质的活性。多重突变可能需要产生戏剧性的遗传后果，如基因失活或产生具有新功能的等位基因。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，我们发现在甲基甲烷磺酸盐（MMS）存在下生长的酵母中可以发生突变簇。慢性暴露于MMS时，前向突变报告基因URA3和CAN1靠近时（间隔1kb）会联合失活，而当它们间隔85kb时则不会，这表明双突变主要是通过单个局部事件发生的。全基因组测序提高到85%的酵母基因组可以检测到单碱基替换的水平（与Piotr Mieczkowski博士合作）。令人惊讶的是，URA3和CAN1的失活通常伴随着额外的突变（多达30个），在跨度高达250 kb的簇中。聚类密度高达1/kb。与基因组其他部分的突变不同，簇主要由G:C对突变组成，并且包含与mms损伤的ssDNA恢复过程中容易出错的TLS突变谱一致的链偏倚。这种碱基特异性和链偏倚表明，DSB相关的链切除是野生型细胞中LHM的主要途径。我们还发现了第二种途径，在缺乏tof1/ timless -csm3/ tippin复制叉保护复合体的突变细胞中产生的ssDNA中发生突变簇。这些较小的集群（仅跨越几个kB）可能源于损坏或未耦合的复制分叉。因此，我们确定了损伤诱导诱变的两种途径，其中局部无法修复DNA损伤和易出错的翻译合成相结合，导致单代内局部严重的遗传改变。这种情况可能导致适应性进化中的快速多样化和选择优势。它还发现了一种可能的遗传疾病和癌症的新来源。我们通过对几十种不同肿瘤的全基因组测序发现的突变进行分析，揭示了三种类型的人类癌症（多发性骨髓瘤、前列腺癌和头颈部鳞状细胞癌）中同时存在的突变簇。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。这些数据表明，通过随机形成的ssDNA的多个同时变化产生的超突变是一种普遍现象，可能是在癌症和正常体细胞组织中产生快速遗传变异的重要机制。