[Preparation of photoreactive oligonucleotide duplexes and their application for photoaffinity modification of DNA-binding proteins]

Bioorg Khim. May-Jun 2001;27(3):205-9. doi: 10.1023/a:1011381304702.
[Article in Russian]

Abstract

To introduce photoreactive dNTP residues to the 3'-end of a mononucleotide gap, base-substituted photoreactive deoxynucleoside triphosphate derivatives, (5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-trans-3-aminopropenyl-1]- and 5-(N-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]- trans-3-aminopropenyl-1)-2'-deoxyuridine 5'-triphosphates, were used as substrates in the DNA polymerase beta-catalyzed reaction. The resulting nick, containing a modified base at the 3'-end, was sealed by T4 phage DNA ligase. This approach enables the preparation of DNA duplexes bearing photoreactive groups at predetermined position(s) of the nucleotide chain. Using the generated photoreactive DNA duplexes, the photoaffinity modifications of DNA polymerase beta and human replicative protein A (hRPA) were carried out. It was shown that DNA polymerase beta and hRPA subunits were modified with the photoreactive double-stranded DNA considerably less effectively than by the nicked DNA. In the case of double-stranded DNA, the hRPA p70 subunit was preferentially labeled, implying a crucial role of this subunit in the protein-DNA interaction.

MeSH terms

  • DNA Polymerase beta / chemistry
  • DNA-Binding Proteins / chemistry*
  • Humans
  • Oligonucleotides / chemistry*
  • Photoaffinity Labels
  • Photochemistry
  • Protein Binding
  • Replication Protein A

Substances

  • DNA-Binding Proteins
  • Oligonucleotides
  • Photoaffinity Labels
  • RPA1 protein, human
  • Replication Protein A
  • DNA Polymerase beta