Understanding how cells repair DNA double-strand breaks (DSBs) is pivotal for genomic stability and preventing mutations associated with diseases like cancer. A recent study by scientists at the Muséum National d’Histoire Naturelle sheds light on the dynamic roles of two key DNA repair pathways—canonical non-homologous end joining (cNHEJ) and microhomology-mediated end joining (MMEJ)—during embryonic development of zebrafish.
DSBs are the most harmful type of DNA damage, potentially leading to cell death or oncogenesis if not repaired correctly. Organisms have evolved various mechanisms to mend these breaks, with MMEJ noted for its mutagenic nature as it relies on microhomologies for repair, often resulting in deletions. Conversely, cNHEJ typically repairs DSBs more faithfully but can still introduce minor errors. Understanding their interplay during the sensitive stages of embryonic development is now more important than ever.
Initially, the research team conducted real-time quantitative polymerase chain reactions (RT-qPCR) to analyze gene expression related to both pathways across seven developmental stages, from fertilization to early larval stages. They found significant changes over time; for example, most MMEJ-related genes showed high levels of expression at the earliest stages, gradually declining, whereas cNHEJ-related genes exhibited increased activity during later stages associated with significant developmental processes like somitogenesis.
Further examination revealed regionalized expression patterns, particularly strong signals found in the developing brain and tissues undergoing rapid cell division. These observations suggest enhanced expression of DNA repair genes where cell proliferation occurs, indicating their significance during key developmental transitions.
To decipher their functional contributions, the researchers employed CRISPR/Cas9 technology to produce mutant zebrafish lines deficient for pivotal genes associated with both pathways. Among these, the loss of DNA polymerase theta (Polθ)—an MMEJ key factor—revealed substantial impacts on embryonic survival, highlighting its role beyond mere DNA repair; it is suggested to protect developing embryos from the dangers of DNA damage.
Contrastingly, the researchers noted the absence of nuclear DNA ligase 3 (nLig3) didn't yield severe phenotypic changes during early development, pointing to potential redundancy or compensation from other enzymatic pathways. They also found the loss of ligase 4, fundamental for cNHEJ, resulted only in larval growth defects, emphasizing the unique roles these proteins play.
Crucially, the utilization of ionizing radiation allowed the team to observe the influence of genotoxic stress on gene expression. At 24 hours post-fertilization following exposure to this stress, increased expression of several DNA DSB repair genes was evident, confirming the adaptive transcriptional response as development progresses.
Interestingly, the absence of Polθ significantly altered the mutation spectrum post-DSB repair, with heightened incidences of insertions at the expense of deletions typically associated with MMEJ, indicating its defining role within this repair pathway.
Overall, this study emphasizes the delicate balance and contextual importance of DNA repair pathways during zebrafish development. Future investigations may lead to more insights on their complex roles across different contexts and stress conditions, with the potential to influence therapeutic strategies targeting DNA repair mechanisms.
Researchers affirm, "Our study highlights the dynamic and contextual roles of cNHEJ and MMEJ pathways during zebrafish development," reinforcing the significance of their findings for both evolutionary biology and medical applications.