Double Strand Break Repair And Recombination

双链断裂修复与重组

• 批准号: 8929700
• 负责人: MICHAEL A RESNICK
• 金额: $ 44.47万
• 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/8929700
• 关键词: Address; Alkylation; Appearance; BRCA1 gene; Cells; Chromosomal Breaks; Chromosomal Instability; Chromosomes; Complex; Cyclin B; DNA; DNA Double Strand Break; DNA Repair; Decision Making; Defect; Dependence; Detection; Development; Digestion; Diploid Cells; Disease; Double Strand Break Repair; EXO1 gene; Electrophoresis; Endonuclease I; Event; Excision; Exhibits; Fission Yeast; Genes; Genetic; Genetic Programming; Genetic Recombination; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Human; Human Herpesvirus 4; In Vitro; Individual; Ionizing radiation; Length; Lesion; Light; Malignant Neoplasms; Modeling; Modification; Molecular; Mutagenesis; Mutation; Organism; Pathway interactions; Physiologic pulse; Poly(ADP-ribose) Polymerases; Process; Production; Proteins; Pulsed-Field Gel Electrophoresis; Radiation; Radiation induced double strand break; Resected; Role; Saccharomycetales; Sampling; Signal Transduction; Site; Source; System; Tail; Techniques; Temperature; Time; Yeasts; base; cell type; endodeoxyribonuclease SceI; endonuclease; gel electrophoresis; homologous recombination; in vivo; inhibitor/antagonist; interest; mung bean; mutant; novel; novel strategies; nuclease; prevent; recombinational repair; repaired; telomere; tool; viral DNA

All organisms detect DSBs and correct them efficiently. It is important to fully understand how DSBs and other types of lesions contribute to genome instability. REPAIR OF DSBS IN YEAST. Using budding yeast, we were the first to directly characterize DSBs, their repair and genetic control over 35 years ago. Recently, we modified systems for detection and revealed opportunities to address an early step in repair, resection. Since yeast chromosomes can be displayed as individual bands according to size using pulse-field gel electrophoresis (PFGE), repair of individual chromosomes can be addressed. There was little if any restitution of full size chromosomal molecules in G1 diploid cells following ionizing radiation (IR). However, DSBs induced in G2 cells were rapidly repaired: >90% DSBs within 2 hr. Repair requires RAD50, -51 and -52. A critical early step in DSB repair and genome stability is resection of ends. While many studies with yeast characterized resection at a unique DSB using site-specific endonucleases, it has been a challenge to address end events at random, dirty-ended DSBs. We developed a novel approach to address resection of IR induced DSBs. Circular chromosomes linearized by a single, random DSB migrate as a unique band during PFGE; however, within 10 min the band shifts to slower mobility and by 1 hr the apparent size increases 75 kb. This PFGE-shift was identical in WT, rad52 and rad51 strains. Mung bean nuclease digestion revealed the shift was due to resection. There was 1 to 2 kb resection per DSB end during repair in WT cells. In rad52 cells the resection rate was similar. However, in a rad50 mutant lacking the MRX complex, resection of radiation- and HO-induced DSBs was drastically reduced. As described in another project ES065073-21, we can apply the Pulse-shift approach to indirect DSBs generated during repair of closely opposed SSBs as well as single-strand gaps. Importantly, using a 2-D PFGE modification our approach has allowed us to identify for the first time resection at 1 or 2 ends of a DSB. In light of difficulties of precise assessment of events at random DSBs, we set out to examine resection at a defined DSB following expression of endonuclease I-SceI. This led to development of 2-D PFGE, which is turning out to be useful in addressing many aspects of resection. Using this approach, it is possible to determine whether the in vivo resection giving rise to an intermediate band known as m* is due to limited resection at the 2 ends of a unique DSB or just longer resection at either end. In this way we could establish the role of various repair components in coordinating resection at both ends of a DSB. Using our PFGE approaches, we provide the first system to distinguish resection at 0, 1 or both ends of DSBs. The 0- and 1-end resections predominate in MRX-null, Sae2, and Mre11 nuclease mutants, suggesting new roles for the cancer-related proteins (Ctp1 and MRN in humans) in repair, namely, efficient and coincident/coordinated resection at both ends of a DSB. We proposed that the structural features of the MRX complex are consistent with coincident/coordinated resection being due to an ability to interact with both DSB ends to directly coordinate resection. In the absence of MRX or SAE2, there is loss of coordination of end-resection. Similar to results with a defined DSB, we found resection is a 2-step process: a) initiation of a short, single-strand 3 tail (100 bases) which is determined by the MRX complex and Sae2, and b) processive 5 degradation carried out by Exo1 and Sgs1/DNA2. In sae2 mutants, initiation is reduced dramatically, but this results in only a 2-fold reduction in rate of repair of IR-DSBs and only a modest reduction in survival. Coincident resection at a clean I-SceI-induced break is much less dependent on Mre11 nuclease or Sae2, contrary to strong dependence on MRX complex. These results suggest a differential role for these functions at dirty and clean DSB ends. The observed MRX coordination of end-resection fits very nicely with our earlier finding of a role for MRX in physically holding the ends of a chromosome break in close proximity. Loss of either EXO1 or SGS1 reduces processivity of resection about 2-fold with little effect on DSB repair or survival. However, resection length is severely reduced in an exo1 sgs1 double mutant. Yet, similar to the sae2 mutant, repair of IR-DSBs is only decreased 2-fold and survival remains high, especially as compared to an MRX mutant. Thus, resection appears to be much greater than what is needed for efficient DSB repair and is not rate limiting in overall repair of IR-induced DSBs. Possibly its most important role is providing large ssDNA regions for signaling to prevent cell progression. TELOMERE PROCESSING. Telomere ends of chromosomes can appear as DSBs including 3 protruding ends. Telomere specific proteins can protect the ends from resection, and there have been many studies on resection after telomere uncapping in yeast and human cells. In budding yeast, when telomeres become uncapped in a cdc13-1 mutant at the restrictive temperature, the amount of resection can be large and was important in studying mutagenesis in ssDNA , while in humans the extent of resection is much smaller (<1 kb). Our approach to addressing 1- and 2-end resections and the ability to quantitate length of resection with nucleases provides a useful tool to understanding components that determine resection. We initiated studies to investigate telomere resection on individual chromosomes, multiple chromosomes and the role of nucleases. Using our PFGE shift approach, we are able to address the roles for endcapping protein Cdc13 and Ku in preventing resection of one or both ends of a chromosome, similar to what was described above for a DSB in a circle. This provides unique opportunities to address differences in stability of telomere ends among the chromosomes, the roles of various components in endprocessing (described above) such as Mre11 and Sae2 (Ctp1) and lengths of resection, when combined with in vitro nuclease treatment. We recently established that Ku prevents extensive resection from nearly all telomere ends in the genome. Much of the resection in the absence of Ku is one ended, but our technique allows us to identify resection at both ends as well. There appear to be chromosomal differences in the 1 and 2 end events that is independent of the chromosome size. We anticipate being able to integrate commonalities in processing of very different kinds of DSB ends in the same cells: telomeres, clean and dirty DSBs. GENERALIZING PFGE-SHIFT TO ADDRESS RESECTION IN OTHER ORGANISMS. Prior to our PFGE-shift approach, there were no systems that provided a ready means for rapid quantitation of resection at random DSBs across the genome, although there were several qualitative foci-based approaches (such as RPA or BRDU) to identify resection in a variety of cell types, including human. Given the large impact of relatively small single-strand tails (i.e., a few 100 bases or more), we have begun to develop a 2-D approach that captures the PFGE-shifting effect of ssDNA tails to identify and quantitate resected molecules across the genome in other organisms without using a circular chromosome. This would provide opportunities to examine components of endprocessing as well as pathway decision-making via resection such as end-joining or recombinational repair. This approach is being applied to fission yeast and eventually to human cells.

所有生物都检测到DSB并有效地纠正它们。重要的是要充分了解DSB和其他类型的病变如何促进基因组不稳定性。 维修DSB在酵母中。使用芽酵母，我们是第一个直接表征DSB的人，即35年前其修复和遗传控制。最近，我们修改了用于检测的系统，并揭示了解决修复，切除的早期步骤的机会。由于使用脉冲场凝胶电泳（PFGE），可以根据尺寸显示酵母染色体作为单个条带，因此可以解决单个染色体的修复。在电离辐射（IR）后，G1二倍体细胞中的全尺寸染色体分子几乎没有恢复。但是，在G2细胞中诱导的DSB迅速修复：> 90％DSB在2小时内。维修需要RAD50，-51和-52。 DSB修复和基因组稳定性的关键早期步骤是末端的切除。尽管许多对酵母菌的研究都使用特定于位点的核酶进行了独特的DSB的切除术，但以随机的，肮脏的DSB来解决最终事件是一个挑战。我们开发了一种新的方法来解决IR诱导的DSB的切除。由单个，随机的DSB线性化的圆形染色体在PFGE期间以独特的频带迁移；但是，在10分钟内，频带转移到较慢的迁移率，到1小时，表观尺寸增加了75 kb。该PFGE换档在WT，RAD52和RAD51菌株中相同。绿豆核酸酶消化揭示了这种转移是由于切除而引起的。在WT细胞中修复过程中，每个DSB末端有1到2 kb的切除术。在Rad52细胞中，切除率相似。然而，在缺乏MRX复合物的Rad50突变体中，辐射和HO诱导的DSB的切除大大降低。如另一个项目ES065073-21中所述，我们可以将脉冲移动方法应用于在紧密相对的SSB和单链间隙的修复过程中产生的间接DSB。 重要的是，使用2-D PFGE修改我们的方法使我们能够在DSB的1或2端首次切除。鉴于对随机DSB的事件进行精确评估的困难，我们着手检查核酸内切酶I-SCEI后定义的DSB的切除。这导致了2-D PFGE的发展，事实证明，这对于解决切除的许多方面很有用。使用这种方法，可以确定导致称为M*的中间带的体内切除是由于独特DSB的2端的切除术有限，还是在任何一端切除更长的切除术。通过这种方式，我们可以确定各种维修组件在DSB两端协调切除方面的作用。使用我们的PFGE方法，我们提供了第一个在DSB的0、1或两端区分切除的系统。 0和1端切除术在MRX-NULL，SAE2和MRE11核酸酶突变体中占主导地位，这表明在DSB的两端，与癌症相关蛋白（人类中的CTP1和MRN在人类中）的新作用（人类在人类中）的作用。我们提出，MRX复合物的结构特征与复合/协调的切除是一致的，这是由于能够与两个DSB末端相互作用以直接协调切除的能力。在没有MRX或SAE2的情况下，最终切除的协调丧失。与定义的DSB的结果相似，我们发现切除是一个2步的过程：a）启动由MRX复合物和SAE2确定的短，单链3尾（100个碱基），以及b）处理用5降解由exo1和sgs1/sgs1/sgs1/dna2进行。在SAE2突变体中，起始大大降低，但这仅导致IR-DSB的修复率降低了2倍，而存活率仅降低。在干净的I-SCEI诱导的断裂下的重合切除率依赖于MRE11核酸酶或SAE2的依赖性要小得多，这与对MRX复合物的强依赖性相反。这些结果表明，在肮脏和清洁的DSB末端，这些功能的作用具有不同的作用。观察到的MRX协调终端分离非常吻合，这与我们早期发现MRX在物理上保持染色体断裂的末端的作用非常吻合。 EXO1或SGS1的丧失可降低切除的加工性约2倍，对DSB修复或存活几乎没有影响。但是，在EXO1 SGS1双突变体中，切除长度大大降低。然而，与SAE2突变体类似，IR-DSB的修复仅降低了2倍，而存活率仍然很高，尤其是与MRX突变体相比。因此，切除似乎比有效的DSB修复所需的切除要大得多，并且在IR诱导的DSB的总体修复中不会限制速率。它最重要的作用可能是提供大型ssDNA区域以防止细胞进展。 端粒处理。染色体的端粒末端可以显示为DSB，包括3个突出末端。端粒特异性蛋白可以保护末端免受切除术，并且在酵母和人类细胞中端粒解膜后切除有许多研究。在萌芽的酵母中，当端粒在限制性温度下在cdc13-1突变体中脱离时，切除量可能很大，对于研究ssDNA中的诱变很重要，而在人类中，切除的程度较小（<1 kb）。我们解决1-和2端切除术的方法以及用核酸酶定量切除长度的能力，为理解确定切除的组件提供了有用的工具。我们启动了研究以研究对单个染色体，多个染色体和核酸酶作用的端粒切除术。使用我们的PFGE偏移方法，我们能够解决终端盖蛋白CDC13和KU在防止染色体切除一端或两端的作用，类似于上面在圆圈中描述的DSB。这为解决染色体之间端粒末端稳定性的差异提供了独特的机会，当与体外核酸酶处理结合时，各种组成部分在末端处理（如上所述）中的作用（如上所述），例如MRE11和SAE2（CTP1）（CTP1）（CTP1）（CTP1）以及切除长度。我们最近确定KU防止了基因组中几乎所有端粒末端的广泛切除。 在没有KU的情况下，大部分切除都是结束的，但是我们的技术使我们也可以在两端确定切除。 在1和2最终事件中似乎存在与染色体大小无关的染色体差异。我们预计能够整合在非常不同类型的DSB中的共同点，以相同的单元格结束：端粒，干净和肮脏的DSB。 概括pFGE换档以解决其他生物的切除术。在我们的PFGE换档方法之前，没有系统能够在整个基因组中快速定量在随机DSB上快速定量切除的方法，尽管有几种基于定性的焦点方法（例如RPA或BRDU）来识别包括人类在内的各种细胞类型（包括人类）的切除。鉴于相对较小的单链尾巴的影响很大（即几百碱基或更多碱基），我们已经开始开发一种二维方法，该方法可以捕获ssDNA尾巴的PFGE转移效应，以识别和定量其他生物体中基因组跨基因组的分子，而无需使用圆形染色体。这将为通过切除（例如最终结合或重组维修）进行检查以及途径决策的组件以及途径决策的机会。这种方法应用于裂变酵母，最终应用于人类细胞。