A new binary system for photosensitized labeling of DNA polymerases in nuclear extract.
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.
Lebedeva
N A
NA
Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia. nataleb@niboch.nsc.ru
Rechkunova
N I
NI
Dezhurov
S V
SV
Khodyreva
S N
SN
Favre
A
A
Lavrik
O I
OI
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology*; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000737:chemistry / Q000378:metabolism; D008297:Male; D015394:Molecular Structure; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019920:Photoaffinity Labels / Q000737:chemistry*; D013737:Testis / Q000201:enzymology; D014020:Tissue Extracts
C119021:5-(N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)aminopropenyl-1)-2'-deoxyuridine-5'-triphosphate; C119022:5-(N-(4-(1-pyrenyl)butylcarbonyl)aminopropenyl-1)-2'-deoxyuridine-5'-triphosphate; C483499:5-(N-(N-(4-azido-2,5-difluoro-3-chloropyridin-6-yl)-3-aminopropionyl)-3-aminopropen-1-yl)-2'-deoxyuridine-5'-triphosphate; D003856:Deoxyuracil Nucleotides; D009687:Nuclear Proteins; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D019920:Photoaffinity Labels; D014020:Tissue Extracts; D004259:DNA-Directed DNA Polymerase