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

N A Lebedeva; N I Rechkunova; S V Dezhurov; S N Khodyreva; A Favre; O I Lavrik

doi:10.1023/a:1023616400794

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.

Authors

N A Lebedeva¹, N I Rechkunova, S V Dezhurov, S N Khodyreva, A Favre, O I Lavrik

Affiliation

¹ Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia. nataleb@niboch.nsc.ru

PMID: 12765532
DOI: 10.1023/a:1023616400794

Abstract

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

Substances

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