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Related Concept Videos

Translesion DNA Polymerases02:10

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...
Proofreading01:31

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
Proofreading01:43

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 Synthesis01:59

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...
Bacterial RNA Polymerase00:43

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...
Bacterial RNA Polymerase00:43

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...

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DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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Evolving a polymerase for hydrophobic base analogues.

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
PubMed
Summary

Researchers developed a new DNA polymerase, 5D4, using directed evolution to efficiently replicate hydrophobic base analogues (HBAs). This breakthrough expands the potential of nucleic acid chemistry and coding for novel applications.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Hydrophobic base analogues (HBAs) offer expanded chemical and coding potential for nucleic acids.
  • However, HBAs are typically poor substrates for DNA polymerases, hindering their application.
  • Discovering HBAs with favorable substrate properties has been a significant challenge.

Purpose of the Study:

  • To develop a strategy for improving HBA substrate properties by directed evolution of a DNA polymerase.
  • To utilize compartmentalized self-replication (CSR) for selecting polymerases capable of replicating specific HBAs.
  • To identify a polymerase with enhanced ability to utilize a range of HBAs.

Main Methods:

  • Directed evolution of chimeric DNA polymerases derived from Thermus genus.
  • Compartmentalized self-replication (CSR) using 5-nitroindole (d5NI) and 5-nitroindole-3-carboxamide (d5NIC) as selection substrates.
  • Characterization of polymerase activity, substrate specificity, and fidelity using biochemical assays and NMR spectroscopy.

Main Results:

  • Isolated a novel DNA polymerase, 5D4, with a broadly enhanced ability to utilize HBAs.
  • 5D4 efficiently formed and extended d5NI and d5NIC self-pairs and heteropairs with all standard bases.
  • The polymerase 5D4 demonstrated activity with various HBA pairs, bypassed diverse HBAs, and enabled PCR amplification of HBA-containing primers with high fidelity.

Conclusions:

  • The directed evolution approach successfully yielded a polymerase (5D4) with significantly improved HBA utilization.
  • 5D4 expands the repertoire of nucleobase analogues amenable to replication and DNA synthesis.
  • This engineered polymerase holds promise for creating nucleic acid polymers with expanded chemical and functional diversity.