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Nucleic Acids Research, 2002, Vol. 30, No. 14 e73
© 2002 Oxford University Press

Binary system for selective photoaffinity labeling of base excision repair DNA polymerases

Olga I. Lavrik1,2, Dmitry M. Kolpashchikov2, Rajendra Prasad1, Robert W. Sobol1 and Samuel H. Wilson*,1

1 Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709, USA and 2 Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 630090 Novosibirsk, Russia

*To whom correspondence should be addressed. Tel: +1 919 541 3267; Fax: +1 919 541 3592; Email: wilson5{at}niehs.nih.gov

A system of photoaffinity 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 fibroblast (MEF) cellular extract in the presence of base-substituted 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 ß 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.


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