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...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-01-01
|
| Series: | Biomolecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2218-273X/15/1/150 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832588913358667776 |
|---|---|
| author | 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 |
| author_facet | 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 |
| author_sort | Colette B. Rogers |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-872ee6a3023e4dffa462a0b89872a7c9 |
| institution | Kabale University |
| issn | 2218-273X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj-art-872ee6a3023e4dffa462a0b89872a7c92025-01-24T13:25:22ZengMDPI AGBiomolecules2218-273X2025-01-0115115010.3390/biom15010150Cell Type Specific Suppression of Hyper-Recombination by Human RAD18 Is Linked to Proliferating Cell Nuclear Antigen K164 UbiquitinationColette B. Rogers0Wendy Leung1Ryan M. Baxley2Rachel E. Kram3Liangjun Wang4Joseph P. Buytendorp5Khoi Le6David A. Largaespada7Eric A. Hendrickson8Anja-Katrin Bielinsky9Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USADepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USADepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USADepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USADepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USADepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USADepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USADepartments of Pediatrics and Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USADepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USADepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USARAD18 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.https://www.mdpi.com/2218-273X/15/1/150gap-fillinghyper-recombinationPCNA K164RAD18ubiquitination |
| spellingShingle | 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 Cell Type Specific Suppression of Hyper-Recombination by Human RAD18 Is Linked to Proliferating Cell Nuclear Antigen K164 Ubiquitination Biomolecules gap-filling hyper-recombination PCNA K164 RAD18 ubiquitination |
| title | Cell Type Specific Suppression of Hyper-Recombination by Human RAD18 Is Linked to Proliferating Cell Nuclear Antigen K164 Ubiquitination |
| title_full | Cell Type Specific Suppression of Hyper-Recombination by Human RAD18 Is Linked to Proliferating Cell Nuclear Antigen K164 Ubiquitination |
| title_fullStr | Cell Type Specific Suppression of Hyper-Recombination by Human RAD18 Is Linked to Proliferating Cell Nuclear Antigen K164 Ubiquitination |
| title_full_unstemmed | Cell Type Specific Suppression of Hyper-Recombination by Human RAD18 Is Linked to Proliferating Cell Nuclear Antigen K164 Ubiquitination |
| title_short | Cell Type Specific Suppression of Hyper-Recombination by Human RAD18 Is Linked to Proliferating Cell Nuclear Antigen K164 Ubiquitination |
| title_sort | cell type specific suppression of hyper recombination by human rad18 is linked to proliferating cell nuclear antigen k164 ubiquitination |
| topic | gap-filling hyper-recombination PCNA K164 RAD18 ubiquitination |
| url | https://www.mdpi.com/2218-273X/15/1/150 |
| work_keys_str_mv | AT colettebrogers celltypespecificsuppressionofhyperrecombinationbyhumanrad18islinkedtoproliferatingcellnuclearantigenk164ubiquitination AT wendyleung celltypespecificsuppressionofhyperrecombinationbyhumanrad18islinkedtoproliferatingcellnuclearantigenk164ubiquitination AT ryanmbaxley celltypespecificsuppressionofhyperrecombinationbyhumanrad18islinkedtoproliferatingcellnuclearantigenk164ubiquitination AT rachelekram celltypespecificsuppressionofhyperrecombinationbyhumanrad18islinkedtoproliferatingcellnuclearantigenk164ubiquitination AT liangjunwang celltypespecificsuppressionofhyperrecombinationbyhumanrad18islinkedtoproliferatingcellnuclearantigenk164ubiquitination AT josephpbuytendorp celltypespecificsuppressionofhyperrecombinationbyhumanrad18islinkedtoproliferatingcellnuclearantigenk164ubiquitination AT khoile celltypespecificsuppressionofhyperrecombinationbyhumanrad18islinkedtoproliferatingcellnuclearantigenk164ubiquitination AT davidalargaespada celltypespecificsuppressionofhyperrecombinationbyhumanrad18islinkedtoproliferatingcellnuclearantigenk164ubiquitination AT ericahendrickson celltypespecificsuppressionofhyperrecombinationbyhumanrad18islinkedtoproliferatingcellnuclearantigenk164ubiquitination AT anjakatrinbielinsky celltypespecificsuppressionofhyperrecombinationbyhumanrad18islinkedtoproliferatingcellnuclearantigenk164ubiquitination |