通过单分子DNA聚合酶I (克莱诺片段) 纳米电路逐步纳入脱氧核三酸盐类似物
Kaitlin M Pugliese1, O Tolga Gul1, Yongki Choi1
1Departments of †Chemistry, §Physics and Astronomy, and ⊥Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.
Journal of the American Chemical Society
|July 7, 2015
概括
DNA聚合酶可以结合改性脱氧核酸三酸盐 (dNTP),单分子研究揭示了基体结合过程中的酶动态. 这表明新生基对的动态稳定性检查机制.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 纳米技术 纳米技术
背景情况:
- DNA聚合酶具有精确识别脱氧核酸三酸盐 (dNTP) 的复杂机制.
- 尽管如此,它们对各种dNTP类似物表现出了显著的耐受性.
- 在分子水平上了解这种耐受性对于DNA复制和修复研究至关重要.
研究的目的:
- 为了研究与dNTP类似物相互作用的DNA聚合酶I克莱诺片段 (KF) 的单分子动力学.
- 通过单壁碳纳米管场效应晶体管 (SWCNT-FETs) 来解决酶对这些类型的适应.
- 阐明基因聚合酶对修饰dNTPs的耐受性背后的机制.
主要方法:
- 将单个KF分子连接到SWCNT-FET以实时进行单分子电流测量.
- 分析本地和模拟dNTPs的基础整合期间的当前变化 (tclosed和topen).
- 利用纳米电路在聚合过程中检测替代酶构成.
主要成果:
- 封闭状态 (τclosed) 的持续时间不受dNTP模拟整合的影响.
- dNTP模拟整合的速度主要受到开放状态持续时间 (τopen) 的影响,反映了分子识别.
- 具体的类似物 (α-thio-dNTPs,6-Cl-2APTP,2-thio-dTTP,2-thio-dCTP) 被纳入的速度较慢,并诱导了替代的酶构造.
结论:
- DNA聚合酶KF对dNTP类型具有耐受性,识别 (τopen) 是限制速度的步骤.
- 该酶利用一个动态稳定性检查机制,涉及O-螺旋旋转,以评估新生的基对稳定性.
- 这种机制,加上全效应,允许容纳非原生基对,不同于以前的模型将所有事件归因于酶关闭.
相关概念视频
The Replisome
39.7K
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...
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...
39.7K
Translesion DNA Polymerases
11.8K
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...
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...
11.8K
Proofreading
9.9K
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.
Errors During Replication are Corrected by the DNA Polymerase...
Errors During Replication are Corrected by the DNA Polymerase...
9.9K
Proofreading
62.3K
Overview
62.3K
Restarting Stalled Replication Forks
6.6K
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,...
6.6K
Lagging Strand Synthesis
64.2K
During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
64.2K


