Base Excision Repair in Early Zebrafish Development: Evidence for DNA Polymerase Switching and Standby AP Endonuclease Activity

The base excision repair (BER) pathway recognizes and repairs most nonbulky lesions, uracil and abasic (AP) sites in DNA. Several participants are embryonic lethals in knockout mice. Since the pathway has never been investigated during embryogenesis, we characterized the first three steps of 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 extracts from all stages are capable of performing BER. Before 3 days postfertilization (dpf), aphidicolin-sensitive polymerases perform most nucleotide insertion. In fact, eggs and early stage embryos lack DNA polymerase-beta protein. After the eggs have hatched at 3 dpf, an aphidicolin-resistant polymerase, probably DNA polymerase-beta, becomes the primary polymerase. Previously, we showed that when the 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 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 stage embryos and adults are the absence of DNA polymerase-beta, leading to predominance of replicative polymerases, and the presence of backup Mg2+-dependent endonuclease activity in early stage embryos. The switch to normal, adult BER occurs fully when the embryos hatch from the chorionic membrane and encounter normal oxidative stress.