几种DNA聚合酶的活性位点上的多种分子相互作用:非极性核酸异构体作为探针
1Contribution from the Department of Chemistry, University of Rochester, Rochester, New York 14627.
Journal of the American Chemical Society
|October 1, 2010
概括
这项研究探讨了DNA聚合酶和逆转录酶与非极性核糖类相似物F,Z和Q的相互作用. 不同的酶表现出不同的能力来复制这些不自然的基对,强调了小沟相互作用的重要性.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 酶学 是一种酶学.
背景情况:
- DNA聚合酶和逆转录酶对于DNA复制和修复至关重要.
- 了解它们与非天然核酸类型的相互作用,可以了解酶机制.
- 之前的研究表明,克莱诺碎片复制了Z-F基对,尽管缺乏沃森-克里克H结合.
研究的目的:
- 通过使用各种DNA聚合酶和逆转录酶,研究与非极性核酸同位素F,Z和Q的蛋白质-DNA相互作用.
- 用这些非自然的基对来描述不同酶的复制效率和选择性.
- 阐明小沟相互作用在聚合酶活性中的作用.
主要方法:
- 对七种不同的DNA聚合酶和逆转录酶的调查.
- 使用的非极性核酸同位素F (提米丁类比物) 和Z,Q (脱氧腺素类比物).
- 评估非自然基对合成的复制效率和选择性,包括T7DNA聚合酶的稳定状态动力学.
主要成果:
- 外核酶无活性的T7DNA聚合酶 (T7(-),水族的DNA聚合酶 (Taq) 和HIV逆转录酶 (HIV-RT) 有效合成了A-F,F-A,F-Z和Z-F基对.
- 牛胸腺DNA聚合酶α (Polα) 和禽髓母细胞瘤病毒逆转录酶 (AMV-RT) 复制了A-F和F-A,但没有F-Z/Z-F;F-Z/Z-F的复制恢复了Q,表明小槽H结合.
- 人类DNA聚合酶β (Pol β) 和莫洛尼小鼠白血病病毒逆转录酶 (MMLV-RT) 无法复制F-Z/Z-F,Q也无法恢复插入,这表明模板和三位点H键是必要的.
结论:
- 在初始基对的DNA小沟相互作用中的酶特异变异对于聚合酶活性至关重要.
- 复制非自然基对的能力取决于特定的聚合酶及其活性位点特征.
- 这项研究证明了结和小沟相互作用在不同酶的DNA合成中的多样性作用.
相关概念视频
Labeling DNA Probes
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Proofreading
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 Enzyme
Errors During Replication are Corrected by the DNA Polymerase Enzyme
Proofreading
Synthesis of new DNA molecules starts when DNA polymerase links nucleotides together in a sequence that is complementary to the template DNA strand. DNA polymerase has a higher affinity for the correct base to ensure fidelity in DNA replication. The DNA polymerase furthermore proofreads 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 EnzymeGenomic DNA is synthesized in...
Translesion DNA Polymerases
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...
Nucleic Acid Structure
The pentose sugar in DNA is deoxyribose, while in RNA the pentose sugar is ribose. The difference between the sugars is the presence of the hydroxyl group on the ribose's second carbon and a hydrogen on the deoxyribose's second carbon. The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms a 5′ to 3′ phosphodiester linkage.
DNA Structure
DNA has a double-helix structure. The...
DNA Structure
DNA has a double-helix structure. The...
DNA Helicases
DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...


