Crystallization and preliminary X-ray analysis of human endonuclease 1 (APE1) in complex with an oligonucleotide containing a 5,6-dihydrouracil (DHU) or an alpha-anomeric 2'-deoxyadenosine (alphadA) modified base

Abstract

The multifunctional human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a key enzyme involved in both the base-excision repair (BER) and nucleotide-incision repair (NIR) pathways. In the NIR pathway, APE1 incises DNA 5' to a number of oxidatively damaged bases. APE1 was crystallized in the presence of a 15-mer DNA containing an oxidatively damaged base in a single central 5,6-dihydrouracil (DHU).T or alpha-anomeric 2'-deoxyadenosine (alphadA).T base pair. Diffraction data sets were collected to 2.2 and 2.7 A resolution from DNA-DHU-APE1 and DNA-alphadA-APE1 crystals, respectively. The crystals were isomorphous and contained one enzyme molecule in the asymmetric unit. Molecular replacement was performed and the initial electron-density maps revealed that in both complexes APE1 had crystallized with a degradation DNA product reduced to a 6-mer, suggesting that NIR and exonuclease reactions occurred prior to crystallization.

Figure 1

Crystals of DNA-DHU–APE1 (a) and DNA-αdA-APE1 (b) (the scale bar is labelled in mm).

Figure 2

Diffraction pattern of the DNA-DHU–APE1 crystal. (a) The outer resolution shell is 2.2 Å (1.7 Å at the edge of the detector). (b) Enlarged view of the region marked by a rectangle in (a).