A new binary system for photosensitized labeling of DNA polymerases in nuclear extract

Biochemistry (Mosc). 2003 Apr;68(4):476-81. doi: 10.1023/a:1023616400794.


A binary system of reagents was used for photosensitized labeling of proteins of bovine testis nuclear extract. A dUTP analog containing 4-azido-2,5-difluoro-3-chloropyridyl group (FAP-dUTP) was used for the first time as a component of the binary system, and a dUTP analog containing the pyrenyl group (Pyr-dUTP) was used as a photosensitizer. Photoaffinity labeling of proteins of nuclear extract was performed using the radioactively labeled DNA duplex with the photoreactive FAP group at the 3;-end of elongating DNA strand and analog of the deoxyribose phosphate residue (3-hydroxy-2-hydroxymethyltetrahydrofuran (F) 5;-phosphate) at the 5;-end of the nick. Such structure is formed by the action of nuclear extract enzymes from the initial DNA duplex containing a synthetic apurine/apyrimidine site and is a photoreactive analog of a long-patch base excision repair intermediate. UV-irradiation modified a limited number of proteins of the nuclear extract. As shown using specific antibodies, the new binary system of photoreagents increases the efficiency of DNA polymerase beta labeling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cattle
  • Cell Nucleus / enzymology*
  • DNA Repair
  • DNA Replication
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyuracil Nucleotides / chemistry
  • Deoxyuracil Nucleotides / metabolism
  • Male
  • Molecular Structure
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism
  • Nucleic Acid Heteroduplexes / chemistry
  • Nucleic Acid Heteroduplexes / genetics
  • Nucleic Acid Heteroduplexes / metabolism
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / genetics
  • Oligodeoxyribonucleotides / metabolism
  • Photoaffinity Labels / chemistry*
  • Testis / enzymology
  • Tissue Extracts


  • 5-(N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)aminopropenyl-1)-2'-deoxyuridine-5'-triphosphate
  • 5-(N-(4-(1-pyrenyl)butylcarbonyl)aminopropenyl-1)-2'-deoxyuridine-5'-triphosphate
  • 5-(N-(N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl)-3-aminopropen-1-yl)-2'-deoxyuridine-5'-triphosphate
  • Deoxyuracil Nucleotides
  • Nuclear Proteins
  • Nucleic Acid Heteroduplexes
  • Oligodeoxyribonucleotides
  • Photoaffinity Labels
  • Tissue Extracts
  • DNA-Directed DNA Polymerase