开发一种聚合酶,用于疏水基类似物.
David Loakes1, José Gallego, Vitor B Pinheiro
1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom.
Journal of the American Chemical Society
|September 26, 2009
概括
研究人员开发了一种新的DNA聚合酶5D4,使用定向进化来有效地复制疏水基类似物 (HBA). 这一突破扩大了核酸化学和编码新应用的潜力.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 合成生物学 合成生物学
背景情况:
- 疏水基类似物 (HBA) 为核酸提供了扩大的化学和编码潜力.
- 然而,HBA通常是DNA聚合酶的不良基质,阻碍了它们的应用.
- 发现具有有利基质特性的HBA一直是一个重大挑战.
研究的目的:
- 通过对DNA聚合酶的定向进化,开发一种改善HBA基质特性的策略.
- 利用分隔自我复制 (CSR) 来选择能够复制特定HBA的聚合酶.
- 为了确定一个聚合酶,提高了利用一系列HBAs的能力.
主要方法:
- 来自Thermus属的仿真DNA聚合酶的定向进化.
- 区分自我复制 (CSR) 使用5 - 英 (d5NI) 和5 - 英-3 - 胺 (d5NIC) 作为选择基质.
- 用生物化学试验和NMR光谱学对聚合酶活性,基质特异性和忠实性的表征.
主要成果:
- 隔离了一种新的DNA聚合酶,5D4,具有广泛增强利用HBAs的能力.
- 5D4有效地形成和扩展d5NI和d5NIC自对和异对,具有所有标准基.
- 聚合酶5D4与各种HBA对表现出活性,绕过各种HBA,并使PCR放大含HBA的原料具有高保真度.
结论:
- 定向进化方法成功产生了聚合酶 (5D4),显著改善了HBA利用率.
- 5D4扩展了核基相似物适合复制和DNA合成的目录.
- 这种工程聚合酶有望创造具有扩大化学和功能多样性的核酸聚合物.
相关概念视频
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...
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...
Lagging Strand Synthesis
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...
Bacterial RNA Polymerase
Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
Bacterial RNA Polymerase
Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...


