Functional evidence for polymerase switching in base excision repair by early zebrafish embryos

Abstract

The base excision repair (BER) pathway recognizes and repairs most non‐bulky lesions, uracil and abasic (AP) sites in DNA. Since the pathway has never been investigated during embryogenesis, we characterized BER in zebrafish extracts from unfertilized eggs, embryos at different developmental stages and adults. Using a 45‐mer double stranded substrate with a U/G mispair at position 21, we showed that all extracts are capable of performing BER. Before 3 dpf aphidicolin‐sensitive polymerases perform most nucleotide insertion. After hatching, an aphidicolin‐resistant polymerase, probably DNA polymeraseß, becomes the primary polymerase. Previously we showed that when zebrafish AP endonuclease protein (ZAP1) level is knocked down, embryos cease dividing after the initial phase of rapid proliferation and die without apoptosis shortly thereafter. Nevertheless, extracts from embryos in which ZAP1 has been largely depleted process substrate equally as well as extracts from control embryos. Since apex1 and apex2 are both strongly expressed in early embryos relative to adults, these data indicate that both may play important roles in DNA repair in early development. In brief, the major differences in BER performed by early embryos and adults are the predominance of replicative polymerases and the presence of backup Mg2+‐dependent endonuclease activity in early stage embryos. Supported by funds from the National Institutes of Health 1R03HD045517, the G. Harold and Leila Y. Mathers Foundation, Aid for Cancer Research, and Northeastern University