Pathways and products of base excision DNA repair in <i>Xenopus laevis</i> eggs: contrast with human cell pathways

Endogenous DNA damage is mutagenic and cytotoxic unless removed by base excision DNA repair (BER). BER replaces 2-10 nucleotides in "long patch" (LP-BER) or just one nucleotide in the simpler "short-patch" SP-BER. LP-BER is required for some oxidative lesions. Xenopus laevis egg extracts (HSS) had robust BER of diverse lesions (uracil and various abasic sites). It was surprising to find no evidence for HSS repair of 8-oxoguanine, an important mutagenic lesion. Also unexpected was the independence of HSS BER from DNA polymerase β (Polβ), which typically contributes both DNA repair synthesis and 5' "trimming" to SP-BER. Instead, HSS BER was blocked by aphidicolin inhibiting the replication DNA polymerases δ and ε. Added human Polβ restored full repair, requiring only its 5'-trimming activity, not its DNA polymerase function. Mass-labeled probes revealed that Xenopus BER repair patches were 80%-90% 2-nucleotide (2-nt) LP-BER tracts. Supplementing the low Polβ level in HSS with recombinant enzyme shifted BER toward 1-nt (SP-BER) patches, also influenced by the surrounding sequence. Excision upstream (5') of the lesion occurred in most of the frog BER products. In contrast, human cell (HEK293) extracts had Polβ-dependent BER, with mostly 2-nt repair patches. Human LP-BER was driven by the Fen1 flap endonuclease, but it did not include 5' excision.