Action of human endonucleases III and VIII upon DNA-containing tandem dihydrouracil

We have shown previously that endonuclease III from Escherichia coli, its yeast homolog Ntg1p and E. coli endonuclease VIII recognize single dihydrouracil (DHU) lesions efficiently. However, these enzymes have limited capacities for completely removing DHU, when the lesion is present on duplex DNA as a tandem lesion. A duplex 30-mer (duplex1920) containing tandem DHU lesions at positions 19 and 20 from the 5′ terminus was used as a substrate for human endonuclease III (hNTH) and endonuclease VIII (NEIL1). Two cleavage products, 18β and 19β were formed, when duplex1920 was treated with hNTH. The 18β corresponded to the expected β-elimination product generated from duplex1920, when the 5′-DHU of the tandem DHU was processed by hNTH. Similarly, 19β is the β-elimination product generated, when the 3′-DHU of the tandem DHU was processed by hNTH; 19β thus still contained a DHU lesion at the 3′ terminus. When these hNTH reaction products were further treated with human APE1, a single new product that corresponded to an 18mer was observed. These data suggested that human APE1 can help to process the 3′ terminals following the action of hNTH on DHU lesions. Similarly, when duplex1920 was treated with NEIL1, two cleavage products, 18p and 19p were observed. The 18p and 19p corresponded to the expected β,δ-elimination products derived from NEIL1 induced cleavage at the 5′-DHU and 3′-DHU of the tandem DHU, respectively. The 3′-phosphoryl group present in 18p can be readily removed by T4 polynucleotide kinase (PNK) to yield an 18mer that is suitable for repair synthesis. However, 19p required the participation of both PNK and APE1 to generate the 18mer. Together, we suggest that the processing of DNA-containing tandem DHU lesions, initiated by hNTH and NEIL1 can be channeled into two sub-pathways, the PNK-independent, APE1-dependent and the PNK, APE1-dependent pathways, respectively.