Photoaffinity labeling of the Klenow fragment with 8-azido-dATP.


The photoaffinity compound 8-azido-dATP was used as a probe for the deoxyribonucleoside triphosphate-binding site of the large fragment of DNA polymerase I. Azido-dATP specifically modified a saturable binding site within the Klenow fragment, and each of the four natural deoxyribonucleoside triphosphate substrates competed with labeling at this site in proportion to its binding constant, as previously defined by equilibrium dialysis. Analysis of tryptic peptides after azido-dATP modification revealed five major cross-linking products, which apparently arose from five distinct photoadducts formed near Tyr-766.




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