Mechanisms of DNA Replication, Chromosome Compaction, and Chromosome Unlinking

DNA 复制、染色体压缩和染色体解联机制

• 批准号: 10618506
• 负责人: KENNETH J MARIANS
• 金额: $ 104.41万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10618506
• 关键词: Amino Acid Sequence; Binding Proteins; Cell Cycle; Cell division; Cells; Cellular Stress; Chromatin; Chromatin Loop; Chromosomal Duplication; Chromosome Segregation; Chromosome Structures; Chromosomes; Complex; DNA; DNA Sequence; DNA Topoisomerase IV; DNA biosynthesis; DNA replication fork; DNA-Directed RNA Polymerase; Failure; Genetic Materials; Genetic Transcription; Genome Stability; Genomic Instability; Head; Human; Lead; MCM Protein; Malignant Neoplasms; Molecular Conformation; Mutation; ORC1L gene; Pathway interactions; Play; Proteins; Reaction; Role; System; Time; Topoisomerase; condensin; daughter cell; genetic information; genome integrity; histone modification; insight; next generation; preservation; prevent; reconstitution; repaired; segregation; transmission process

Summary Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. It is now clear that replication forks stall frequently as a result of encounters between the replication machinery and template damage, slow-moving or paused transcription complexes [TC(s)], unrelieved positive superhelical tension, covalent protein-DNA complexes, and as a component of cellular stress responses. Stalled forks are foci for genomic instability that causes genetic alterations and can give rise to cancer. Stalled forks must be protected/remodeled/repaired and replication restarted/continued in order to maintain genomic stability. We propose to continue our analyses of replication fork stalling brought about by such factors. We ask: (i) Are replisome collisions with protein-bound R-loops more prone to stall forks than collisions with unbound R- loops? which are only transient obstacles to progression. (ii) Does replication fork reversal preserve replication potential during replication-transcription conflicts? (iii) Does the accumulation of positive superhelicity between replisomes and TCs approaching each other head-on lead to replication fork stalling and/or collapse? And (iv), how do replisomes overcome collisions with RNA polymerases that are themselves stalled by DNA template damage? We have used the MIRA mechanism to begin to transit our focus on bacterial replication systems to replication with human proteins. We will investigate the DNA sequence requirements for loading of double hexamers of the MCM proteins to DNA, as well as the effect of chromatinization of the DNA substrate on the loading reaction. We ask: (i) what are the requirements for various amino acid sequence motifs in ORC1? (ii) What role is played by ORC6 (we currently do not require this protein for loading)? And (iii) what are the effects of histone modifications in the loading reaction? We are also proceeding to reconstitute the complete replication reaction with purified human replication proteins. Such a system will afford unprecedented insight into insults to replication fork progression and cellular stress responses. Coordinating the structural organization of chromosomes is essential for DNA replication, transcription, and chromosome segregation during cell division. Failure to achieve proper chromosomal organization during separation can result in DNA breakage, leading to an uneven distribution of the genetic material to the next generation. We propose to continue our analyses of the mechanisms by which the bacterial condensin MukBEF and the cellular decatenase topoisomerase IV cooperate to promote proper chromosome compaction and segregation. We ask: (i) Does the MukBEF complex either translocate on or extrude loops of DNA? And (ii) how does replication proceed through topological domains generated by MukB and Topo IV?

总结 遗传信息的准确传递需要染色体DNA的完全复制， 细胞分裂周期现在很清楚，复制分叉经常由于 复制机制和模板损伤，缓慢移动或暂停的转录复合物[TC（s）]， 未解除的正超螺旋张力，共价蛋白质-DNA复合物，并作为细胞的组成部分， 应激反应失速叉是基因组不稳定性的焦点，导致遗传改变， 到癌症必须保护/改造/修复停滞的分叉，并重新启动/继续复制， 保持基因组的稳定性。 我们建议继续分析这些因素导致的复制叉停滞。我们问： (i)复制体与蛋白质结合的R环的碰撞是否比与未结合的R环的碰撞更容易产生失速叉？ 循环？这只是前进的暂时障碍。(ii)复制分叉反转是否保留复制 在复制-转录冲突期间的潜在可能性？(iii)正的超螺旋度的积累 复制体和TC迎面接近会导致复制叉停滞和/或崩溃吗？和（iv）， 复制体是如何克服与RNA聚合酶的碰撞的？ 损坏？ 我们已经使用MIRA机制开始将我们对细菌复制系统的关注转移到 复制人类蛋白质。我们将研究装载双链DNA的DNA序列要求， 的MCM蛋白质的六聚体的DNA，以及DNA底物的染色质化的影响， 加载反应我们问：（i）ORC 1中各种氨基酸序列基序的要求是什么？（二） ORC 6发挥了什么作用（我们目前不需要这种蛋白质进行加载）？及（iii）有何影响 组蛋白的修饰我们也在着手重建 用纯化的人复制蛋白进行复制反应。这样一个系统将提供前所未有的洞察力 转化为对复制叉进展和细胞应激反应的损害。 协调染色体的结构组织对于DNA复制、转录、 和细胞分裂时染色体分离。未能实现适当的染色体组织， 分离会导致DNA断裂，导致遗传物质在下一个群体中的不均匀分布。 一代我们建议继续分析细菌凝聚的机制， MukBEF和细胞decatenase拓扑异构酶IV合作促进适当的染色体压实 和种族隔离我们问：（i）MukBEF复合物是否在DNA环上移位或挤出？和 (ii)复制是如何通过MukB和Topo IV生成的拓扑结构域进行的？