Large Screening And Functional Analysis Of E3Ubiquitin Ligase In DNA Damage Response

Genomic DNA suffered a variety of insults caused by endogenous and environmental factors constantly. Unrepaired or misrepaired lesions would lead to mutations, chromosomal instability, and cell death, which eventually lead to tumorigenesis.Mammalian cells have developed a complicated protective system including DNA repair and cell signaling to retain their genomic integrity upon DNA insults. The mechanism of DDR-associated protein actions on different types of damage always depends on protein post-translational modification (PTM), such as phosphorylation, acetylation, methylation, and ubiquitination, etc. Ubiquitination emerged as an important form of modification that can regulate DNA damage signaling pathways. The ubiquitin signaling cascade involves E1ubiquitin-activating enzyme, E2ubiquitin-conjugating enzyme, and E3ubiquitin-ligase.Recently, more than500E3ligases were identified in humans. To identify unknown components involved in the DNA damage response pathway, we cloned about300E3ligases. Using53BP1ionizing radiation induced foci as a marker, we carried out an immunofluorescence staining screen to identify E3ligases involved in the recruitment of53BP1to DNA damage sites. In this screening, we identified several known DDR-associated proteins (such as RNF4, RNF8, RNF168, PSO4, etc)1-6, confirming that the screening method is reliable. We speculated that some unknown DDR relevant E3ligases might exist among these candidates. We’ve identified several putative E3ligases which might serve as components that contribute to DNA repair.