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Regulated eukaryotic DNA replication origin firing with purified proteins

机译：纯化蛋白调控的真核DNA复制起点

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摘要

能够利用纯化的蛋白从其最初放电(origin firing)的最早阶段来重构一个真核系统的DNA 复制是人们长期希望实现的一个目标。然而，真核生物相对于细菌和噬菌体系统所具有的更大复杂性影响了这一发展。但现在，John Diffley及同事成功地在试管中重构了芽殖酵母DNA复制的最初事件。这一纯化的系统含有42种蛋白(包括16个复合物)，它确定了''依赖于起源的启动"(origin-dependent initiation)的分阶段过程(其中包括激酶对其之调控)。%Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo, Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.
机译：能够利用纯化的蛋白从其最初放电(origin firing)的最早阶段来重构一个真核系统的DNA 复制是人们长期希望实现的一个目标。然而，真核生物相对于细菌和噬菌体系统所具有的更大复杂性影响了这一发展。但现在，John Diffley及同事成功地在试管中重构了芽殖酵母DNA复制的最初事件。这一纯化的系统含有42种蛋白(包括16个复合物)，它确定了''依赖于起源的启动"(origin-dependent initiation)的分阶段过程(其中包括激酶对其之调控)。%Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo, Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

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  • 来源
    《Nature》 |2015年第7544期|431-435q2|共6页
  • 作者

    Joseph T. P. Yeeles; Tom D. Deegan; Agnieszka Janska; Anne Early; John F. X. Diffley;

  • 作者单位

    Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK;

    Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK;

    Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK;

    Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK;

    Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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