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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.
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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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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.
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Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Pulak Ghosh1, Karin Betz2, Cédric Gutfreund2

  • 1Department of Chemistry, Indian Institute of Science Education and Research (IISER), Pune, Dr. Homi Bhabha Road, Pune, 411008, India.

Angewandte Chemie (International Ed. in English)
|October 21, 2024
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Summary
This summary is machine-generated.

DNA polymerases incorporate modified nucleotides, with heterocycle size impacting incorporation. Environment-sensitive probes like FBFdUTP allow real-time monitoring of polymerase activity, aiding next-generation probe development.

Keywords:
19F NMR spectroscopyDNA polymerasesX-ray crystallographyfluorescent nucleotide probesnucleosides

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Nucleic acid functionalization is crucial for biophysical and biotechnology.
  • DNA polymerases play a key role in incorporating modified nucleotides.

Purpose of the Study:

  • To understand DNA polymerase recognition and incorporation of modified nucleotides.
  • To develop advanced nucleotide probes with diverse chemical modifications.
  • To elucidate the structural basis for accommodating modified substrates.

Main Methods:

  • X-ray crystallography of ternary complexes with modified dUTP probes (SedUTP, BFdUTP, FBFdUTP).
  • Single nucleotide incorporation and primer extension assays.
  • Fluorescence and 19F NMR spectroscopy for real-time monitoring.

Main Results:

  • Heterocycle size at the C5 position of uracil influences nucleotide incorporation by DNA polymerases.
  • SedUTP and FBFdUTP function as dual-purpose probes, combining fluorescence with X-ray or 19F NMR labels.
  • FBFdUTP enabled real-time monitoring of polymerase activity.
  • Crystal structures revealed the role of gatekeeper amino acids (Arg660, Arg587) in substrate processing.

Conclusions:

  • DNA polymerase families A and B exhibit plasticity in accommodating modified nucleotides.
  • The study provides structural insights into enzyme-substrate interactions.
  • Findings facilitate the development of novel nucleotide probes for various applications.