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相关概念视频

Translesion DNA Polymerases02:10

Translesion DNA Polymerases

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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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DNA Isolation01:24

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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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Spontaneous mutations arise infrequently during DNA replication due to errors in the process. A key factor behind these errors is tautomeric shifts in nitrogenous bases, where bases transition from keto to enol forms or amino to imino forms. This shift can alter base-pairing rules, leading to mutations. Additionally, reactive oxygen species (ROS) arising from aerobic metabolism can damage DNA, resulting in depurination (loss of a purine base) or depyrimidination (loss of a pyrimidine base).
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Nucleoside Triphosphates - From Synthesis to Biochemical Characterization
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在dUTP C5-替代剂和DNA聚合酶之间非共价相互作用降低PCR效率

Olga A Zasedateleva1, Sergey A Surzhikov1, Viktoriya E Kuznetsova1

  • 1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, 119991 Moscow, Russia.

International journal of molecular sciences
|September 9, 2023
PubMed
概括
此摘要是机器生成的。

分子建模和PCR实验显示,修改后的dUTPs (脱氧氨酸三酸盐) 降低了PCR放大效率. 这种减少与dUTP替代剂和DNA聚合酶之间的非共价键增加有关.

关键词:
A和B家族的DNA聚合酶.修改了C5的dUTPs可以使用.进行PCR放大.在X射线中,X射线结构结构是什么?分子建模分子建模非共价相互作用非共价相互作用

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科学领域:

  • 生物化学 生物化学
  • 分子生物学分子生物学
  • 计算化学计算化学

背景情况:

  • 脱氧核酸三酸盐 (dNTP) 是DNA聚合酶的基本基质.
  • 修改后的dNTPs可以表现出改变的特性,影响聚合酶活性和PCR结果.
  • 了解聚合酶-dNTP相互作用对于开发新型分子工具至关重要.

研究的目的:

  • 调查修改后的dUTPs对PCR放大效率在各种DNA聚合酶的影响.
  • 将实验PCR数据与聚合酶-dNTP相互作用的分子建模预测相关联.
  • 为了确定调节dNTPs在酶性DNA合成中的活性的关键分子特征.

主要方法:

  • 利用分子建模研究具有新型聚合酶特异性特性的脱氧氨酸三酸盐 (dUTPs).
  • 使用修改后的dUTP与一组DNA聚合酶 (Taq,Tth,Pfu,Vent,Deep Vent,Vent (exo-),Deep Vent (exo-)) 进行PCR放大实验.
  • 将实验PCR效率数据与KlenTaq聚合酶-DNA-dNTP复合物的3D结构建模进行比较.

主要成果:

  • 修改后的dUTPs,功能化与重的或芳香的组,被测试PCR放大效率.
  • 观察到PCR效率下降,dUTP替代剂和DNA聚合酶之间的非共价键数量增加.
  • 量化了PCR效率每增加一个非共价键的约15%的下降.
  • 这些发现被推广到A和B家族的DNA聚合酶.

结论:

  • 在dNTP替代剂和DNA聚合酶之间非共价键的数量是PCR效率的关键决定因素.
  • 这种相互作用参数可以调节以调节DNA聚合酶活性.
  • 分子建模提供了一种有价值的方法,用于预测和理解在酶反应中修改后的dNTPs的行为.