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中文摘要
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摘要 遗传信息的准确传递需要完全复制每条染色体的DNA 细胞分裂周期。现在很明显,复制派生经常由于 复制机制和模板损伤、缓慢移动或暂停的转录复合体[TC(S)], 未缓解的正超螺旋张力,共价蛋白质-DNA复合体,并作为细胞的组成部分 压力反应。失速的叉子是基因组不稳定的焦点,这种不稳定会导致基因变化,并可能导致 致癌。必须保护/重建/修复停滞的分叉,并重新启动/继续复制,以便 保持基因组的稳定性。 我们建议继续分析由这些因素造成的复制分叉失速。我们问: (I)与蛋白质结合的R-环的复制体碰撞比与未结合的R-环的碰撞更容易发生失速分叉 循环?这些只是前进的短暂障碍。(Ii)复制分叉反转是否保留复制 潜在的复制-转录冲突?(Iii)正的超螺旋度的积累是否 复制体和TCS相互迎头靠近会导致复制分叉停滞和/或崩溃吗?及(四)、 复制体如何克服与RNA聚合酶的碰撞,而RNA聚合酶本身是被DNA模板阻挡的 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 用人类蛋白质进行复制。我们将调查加载双基因的DNA序列要求 MCM蛋白对DNA的六聚体,以及DNA底物的显色化对 加载反应。我们问:(I)ORC1中对各种氨基酸序列基序的要求是什么?(Ii) ORC6扮演什么角色(我们目前不需要这种蛋白质来加载)?以及(Iii)有何影响? 组蛋白修饰在负载反应中的作用?我们还在着手重建完整的 与纯化的人类复制蛋白发生复制反应。这样的系统将提供前所未有的洞察力 转化为对复制分叉进程和细胞应激反应的侮辱。 协调染色体的结构组织对于DNA复制、转录、 以及细胞分裂过程中的染色体分离。没有达到适当的染色体组织在 分离可能导致DNA断裂,导致遗传物质不均匀地分配给下一个 一代。我们建议继续分析细菌凝结的机制 MukBEF和细胞去酯酶拓扑异构酶IV共同促进适当的染色体紧凑 和种族隔离。我们问:(I)MukBEF复合体是移位在DNA环上还是挤出DNA环?和 (Ii)复制是如何通过MukB和Topo IV产生的拓扑域进行的?
英文摘要
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?
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Mechanisms of DNA Replication, Chromosome Compaction, and Chromosome Unlinking
  • 批准号:
    9900025
  • 项目类别:
  • 资助金额:
    $102.86万
  • 财政年份:
    2018
  • 负责人:
    KENNETH J MARIANS
  • 依托单位:
Mechanisms of DNA Replication, Chromosome Compaction, and Chromosome Unlinking
  • 批准号:
    10373984
  • 项目类别:
  • 资助金额:
    $102.86万
  • 财政年份:
    2018
  • 负责人:
    KENNETH J MARIANS
  • 依托单位:
Topoisomerases and Chromosome Segregation
  • 批准号:
    7988465
  • 项目类别:
  • 资助金额:
    $13.43万
  • 财政年份:
    2009
  • 负责人:
    KENNETH J MARIANS
  • 依托单位:
Integrated PhD Training Program in Cancer Biology
  • 批准号:
    7293596
  • 项目类别:
  • 资助金额:
    $21.81万
  • 财政年份:
    2006
  • 负责人:
    KENNETH J MARIANS
  • 依托单位:
海外基金