Cell Type Specific Suppression of Hyper-Recombination by Human RAD18 Is Linked to Proliferating Cell Nuclear Antigen K164 Ubiquitination

Cell Type Specific Suppression of Hyper-Recombination by Human RAD18 Is Linked to Proliferating Cell Nuclear Antigen K164 Ubiquitination

RAD18 is a conserved eukaryotic E3 ubiquitin ligase that promotes genome stability through multiple pathways. One of these is gap-filling DNA synthesis at active replication forks and in post-replicative DNA. RAD18 also regulates homologous recombination (HR) repair of DNA breaks; however, the curre...

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Main Authors: Colette B. Rogers, Wendy Leung, Ryan M. Baxley, Rachel E. Kram, Liangjun Wang, Joseph P. Buytendorp, Khoi Le, David A. Largaespada, Eric A. Hendrickson, Anja-Katrin Bielinsky
Format: Article
Language:English
Published: MDPI AG 2025-01-01
Series:Biomolecules
Subjects:
gap-filling
hyper-recombination
PCNA K164
RAD18
ubiquitination
Online Access:https://www.mdpi.com/2218-273X/15/1/150
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Summary:RAD18 is a conserved eukaryotic E3 ubiquitin ligase that promotes genome stability through multiple pathways. One of these is gap-filling DNA synthesis at active replication forks and in post-replicative DNA. RAD18 also regulates homologous recombination (HR) repair of DNA breaks; however, the current literature describing the contribution of RAD18 to HR in mammalian systems has not reached a consensus. To investigate this, we examined three independent <i>RAD18</i>-null human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e., HCT116 mutants were hyper-recombinogenic (hyper-rec). Interestingly, these phenotypes were linked to RAD18’s role in PCNA K164 ubiquitination, as HCT116 <i>PCNA<sup>K164R/+</sup></i> mutants were also hyper-rec, consistent with previous studies in <i>rad18</i><sup>−/−</sup> and <i>pcna<sup>K164R</sup></i> avian DT40 cells. Importantly, the knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. We propose that the hierarchy of post-replicative repair and HR, intrinsic to each cell type, dictates whether RAD18 is required for suppression of hyper-recombination and that this function is linked to PCNA K164 ubiquitination.
ISSN:2218-273X

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