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相关概念视频

Replication in Eukaryotes02:31

Replication in Eukaryotes

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Overview
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The Replisome03:01

The Replisome

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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
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DNA Isolation01:24

DNA Isolation

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DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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DNA Replication02:40

DNA Replication

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DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.  After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Replication in Prokaryotes
DNA replication...
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Replication in Prokaryotes01:32

Replication in Prokaryotes

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DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell's circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
Many Proteins Work Together to Replicate the Chromosome
Replication is coordinated and carried out by a host of specialized...
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Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

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For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
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Cryo-EM maps of human DNA polymerase ε should be reevaluated in light of its unexpected behavior in vitro.

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Competition for the nascent leading strand shapes the requirements for PCNA loading in the replisome.

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Structural basis for processive daughter-strand synthesis and proofreading by the human leading-strand DNA polymerase Pol ε.

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相关实验视频

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Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein
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Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein

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使用纯化人类蛋白质进行快速高效的DNA复制

Yasemin Baris1, Martin R G Taylor1, Valentina Aria1

  • 1MRC Laboratory of Molecular Biology, Cambridge, UK.

Nature
|May 18, 2022
PubMed
概括

研究人员重建了人体复制体, 发现DNA聚合酶epsilon (Polε) 是主要链合成的关键. 这一过程需要PCNA和CTF18-RFC,AND-1直接协助主链复制.

科学领域:

  • 分子生物学
  • 遗传学
  • 生物化学

背景情况:

  • 人类复制体对于DNA复制是必不可少的,它包含许多围绕CMG酶组织的蛋白质.
  • 人类复制体从纯化的蛋白质中完全复制是缺乏的, 阻碍了对其精确功能的理解.

研究的目的:

  • 使用纯化成分生化复制功能的人类复合体.
  • 阐明关键蛋白质,包括DNA聚合酶和辅助因子在人类DNA复制中的特定作用.

主要方法:

  • 使用11种纯化的人类复制因子 (43种多) 的人体复制体的生物化学复制.
  • 用于测量DNA复制速度和效率.
  • 研究PCNA,CTF18-RFC,CLASPIN,TIMELESS-TIPIN和AND-1在复制体功能中的作用.

主要成果:

  • 成功重建了能够快速有效地复制DNA的人类复制体.
  • 确定DNA聚合酶epsilon (Polε) 对于最佳的前导链合成至关重要,依赖PCNA和CTF18-RFC.
  • 证明AND-1直接增强领先链的复制,与酵母不同,Polα被独立招募用于滞后链的初始化.

结论:

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  • 重建的人类复制体提供了一个研究DNA复制机制的系统.
  • 揭示了Polε,PCNA,CTF18-RFC和AND-1在人类主要链合成中的不同作用.
  • 澄清了在人类DNA复制中用于滞后链启动的Polα的独立招募.