Sensitized photomodification of mammalian DNA polymerase β. A new approach for highly selective affinity labeling of polymerases (original) (raw)
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A Binary System of Photoreagents for High-Efficiency Labeling of DNA Polymerases
Biochemical and Biophysical Research Communications, 2001
To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioactive primers were synthesized by DNA polymerases beta (pol beta) or DNA polymerase from Thermus thermophilus (pol Tte) using a template-primer duplex in the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl group linked via spacers of varying length to 5-position of uridine ring- 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidobenzoyl)-butanoyl]-amino]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 365-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr- 8-dUTP) or 5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in the case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3'-end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymerases in comparison to the primer crosslinking without photosensitizer.
Highly Efficient Labeling of DNA Polymerases by a Binary System of Photoaffinity Reagents
2002
Thermus thermophilus B35; FAB 4 dUTP) 5 [N (2,3,5,6 tetra fluoro 4 azidobenzoyl) 3 amino trans propenyl 1] 2′ deoxyuridine 5′ triphosphate; FAB 4 ddUTP) 5 [N (2,3,5,6 tetrafluoro 4 azidobenzoyl) 3 amino trans propenyl 1] 2′,3′ dideoxyuridine 5′ triphosphate; FAB 9 dUTP) 5 [N [[(2,3,5, 6 tetrafluoro 4 azidobenzoyl) butanoyl] amino] 3 amino trans propenyl 1] 2′ deoxyuridine 5′ triphosphate; Pyr 8 dUTP) 5 [N (4 (1 pyrenyl) butylcarbonyl) 3 amino trans propenyl 1] 2′ deoxyuridine 5′ triphosphate; Pyr 6 dUTP) 5 [N (4 (1 pyrenyl) ethylcarbonyl) 3 amino trans propenyl 1] 2′ deoxyuridine 5′ triphosphate.
Binary system for selective photoaffinity labeling of base excision repair DNA polymerases
Nucleic Acids Research, 2002
A system of photoaf®nity reagents for selective labeling of DNA polymerases in extracts has been examined. To create the photoreactive DNA probe in situ, DNA substrates containing a synthetic abasic site are incubated in mouse embryonic ®broblast (MEF) cellular extract in the presence of basesubstituted arylazido derivatives of dNTPs. This results in synthesis of a photoreactive long patch base excision repair (BER) intermediate. The arylazido photoreactive group is then activated through energy transfer from the pyrene group of a dNTP analog (Pyr-dUTP), following 365 nm UV light exposure. Pyr-dUTP binds to the active site of DNA polymerases, and the pyrene group, when excited by 365 nm UV light, activates the nearby photoreactive group in the BER intermediate resulting in crosslinking of DNA-bound DNA polymerases. Under these conditions, various DNA binding proteins that are unable to bind Pyr-dUTP are not crosslinked to DNA. DNA polymerase b is the predominant crosslinked protein observed in the MEF extract. In contrast, several other DNA binding proteins are labeled under conditions of direct UV light activation of the photoreactive group at 312 nm. This study illustrates use of a new method of selective labeling of DNA polymerases in a crude cellular extract.
2001
To introduce photoreactive dNMP 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-3aminopropenyl-1]-and 5-{ N-[ N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-trans-3aminopropenyl-1}-2'-deoxyuridine 5'-triphosphates, were used as substrates in the DNA polymerase β-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 a predetermined position of the nucleotide chain. Using the generated photoreactive DNA duplexes, the photoaffinity modifications of DNA polymerase β and human replication protein A (hRPA) were carried out. It was shown that DNA polymerase β 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.
Proceedings of the National Academy of Sciences, 1986
We have synthesized the photoactive deoxyuridine nucleotide 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP) and used it to synthesize light-sensitive DNA by enzymatic incorporation. In the absence of ultraviolet light, 5-N3dUTP is a substrate for Escherichia coli DNA polymerase I. In in vitro DNA synthesis reactions using bacteriophage M13 single-stranded DNA as the template and 5-N3dUTP in place of dTTP, a photoactive complementary strand was synthesized by DNA polymerase I. The complementary strand was not synthesized when the 5-N3dUTP was substituted for dCTP or when it was exposed to ultraviolet light prior to the addition of DNA polymerase I. Using a synthetic lac operator template of 26 bases and a 15-base primer, we generated a photoactive 26-base-pair lac operator by enzymatically incorporating 5-N3dUMP with DNA polymerase I. Crosslinking of this photoactive DNA fragment to lac repressor was totally dependent on the presence of UV light and was reduced 78% by 150 ...
2001
Substrate properties of the earlier synthesized and characterized dCTP derivatives bearing in the exo-N-position of cytosine 2-(4-azido-2,3,5,6-tetrafluorobenzoylamino)ethyl (I), 2-(2-nitro-5-azidobenzoylamino)ethyl (II), 2-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxymethylcarbonylamino)ethyl (III), 4-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy)butyloxy (IV), or 4-(4-azido-2,3,5,6-tetrafluorobenzylidenehydrazinocarbonyl)butylcarbonylamino (V) groups were studied in the primer extension reaction catalyzed by rat DNA polymerase ß. Unlike the earlier results obtained with HIV reverse transcriptase, dCTP derivatives (I)–(III) were not recognized by rat DNA polymerase
Highly selective affinity labeling of the primer-binding site ofE.coliDNA polymerase I
FEBS Letters, 1990
Highly selective affinity labeling of the primer site of E.coli DNA polymerase I was performed with the S-reactive derivatives of oligothymidylate in the presence of poly(dA) template. Subtilysine cleavage proved that the site of affinity modification belonged to the 'Klenow' part of DNA polymerase I. If taken separately, Klenow fragment was not labeled by these oligonucleotide derivatives. The sites of affinity labeling were tested in the structure of DNA polymerase I by hydroxylamine cleavage. At least two sites of labeling were revealed. The main one was localized between Gly-833 and His-928. E.coti DNA polymerase I; Klenow fragment; Affinity labeling 2.4. Cleavage of the affinity labeled DNA polymerase I Cleavage by subtilysine was carried out according to Klenow et al.
Russian Journal of Bioorganic Chemistry, 2000
Abstraet--A new reagent for photoaffinity modification of biopolymers, 5-[E-N-(2-nitro-5-azidobenzoyl)-3amino-l-propen-l-yl]-2',3'-dideoxyuridine 5'-triphosphate (NAB-ddUTP), was synthesized. Like a similar derivative of 2'-deoxyuridine 5'-triphosphate (NAB-dUTP), it was shown to be able to effectively substitute for dTrP in the synthesis of DNA catalyzed by eukaryotic DNA polymerase 1~ and to terminate DNA synthesis. A 5'-32p-labeled primer with a photoreactive group at the 3'-terminus was derived from NAB-ddUTP and used for photoaffinity labeling of the human replication protein A (RPA). The covalent attachment of RPA p32 and p70 subunits to the labeled primers was demonstrated. NAB-ddUTP is a promising tool for studying the interaction of proteins of the replicative complex with NA in cellular extracts and living cells during the termination of DNA synthesis.