Related Experiment Video
Updated: Jun 23, 2026
Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System
Published on: April 30, 2018
Base excision repair in early zebrafish development: evidence for DNA polymerase switching and standby AP
Sean Fortier1, Xiaojie Yang, Yi Wang
1Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Abstract:
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 Mg(2+)-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.
Insights
Zebrafish DNA repair differs in early development, with replicative polymerases and backup endonuclease activity present before hatching. After 3 days postfertilization, DNA polymerase-beta becomes dominant, enabling standard base excision repair (BER).
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- The base excision repair (BER) pathway is crucial for repairing DNA damage like uracil and abasic sites.
- BER pathway components are essential for embryonic development, with some being embryonic lethals in knockout mice.
- The BER pathway's role during embryogenesis has not been previously investigated.
Purpose of the Study:
- To characterize the initial three steps of the BER pathway in zebrafish during embryogenesis.
- To compare BER activity in zebrafish extracts from unfertilized eggs, various embryonic stages, and adult tissues.
- To identify developmental stage-specific differences in BER enzyme utilization.
Main Methods:
- Utilized a 45-mer double-stranded DNA substrate with a uracil/guanine mispair.
- Assayed BER activity in zebrafish extracts from different developmental stages (unfertilized eggs, embryos, adults).
- Investigated the role of aphidicolin-sensitive and resistant polymerases, and AP endonuclease activity.
Main Results:
- Zebrafish extracts from all developmental stages demonstrated BER capability.
- Early embryos (before 3 days postfertilization) primarily used aphidicolin-sensitive polymerases due to the absence of DNA polymerase-beta.
- Post-hatching embryos (after 3 days postfertilization) showed a shift to aphidicolin-resistant polymerases, likely DNA polymerase-beta, as the primary polymerase.
- Despite ZAP1 knockdown affecting embryonic division, BER activity in depleted extracts remained comparable to controls, suggesting roles for apex1 and apex2 in early development.
- Early embryos exhibited backup Mg(2+)-dependent endonuclease activity, which is absent in adults.
Conclusions:
- Zebrafish embryogenesis involves distinct BER mechanisms compared to adults, notably the absence of DNA polymerase-beta and reliance on replicative polymerases.
- The transition to adult-like BER occurs post-hatching, coinciding with increased oxidative stress.
- Apex1 and Apex2 likely play significant roles in DNA repair during early zebrafish development.
Related Concept Videos
01:02Long-patch Base Excision Repair
01:54Base Excision Repair
The first step of...
01:54Base Excision Repair
The first step of...
01:31Proofreading
Errors During Replication are Corrected by the DNA Polymerase Enzyme
01:43Proofreading
02:37Restarting Stalled Replication Forks