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Stimulation of APE1 activity by the pol β complex or purified PARP-1.

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posted on 2015-05-28, 04:29 authored by Rajendra Prasad, Nadezhda Dyrkheeva, Jason Williams, Samuel H. Wilson

A schematic representation of the DNA substrate containing the AP-site analogue THF is shown at the top. The reaction conditions and product analysis are described under Materials and Methods. (A) APE1 incision reactions were assembled on ice either with increasing amounts of pol β complex (A) or with increasing amounts of purified PARP-1 (B) The incision reaction was initiated by addition of 0.1 nM APE1 and transferring the reaction mixtures to 37°C for 10 min. The reaction products were analyzed as in Fig 1. The positions of the 32P-labeled substrate and the product of APE1 strand incision are indicated. A representative phosphorimage of two repeats is illustrated. (C) Quantification of the APE1 product formed at the highest amount of pol β complex (3 μl) and the highest concentration of PARP-1 (50 nM) reveal an approximately 3-fold increase in APE1 activity as compared to that of APE1 alone. The mean of two repeats is illustrated.