Cellular and Molecular Medicine
Mosaic of Interests:
The E3 SUMO Ligase MMS21 Promotes HR through DSB Formation at Reversed Forks
Kelvin W. Pond, Christelle de Renty, Mary K. Yagle, Nathan A. Ellis
Department of Cellular and Molecular Medicine and the University of Arizona Cancer, Tucson, AZ
DNA damage during DNA replication is a frequent event, and can lead to genetic mutations if left unrepaired. Mutations in the genes responsible for the repair of DNA damage increase the rate at which other mutations accumulate in a cell. This is referred to as genomic instability; a major driver of cancer progression. DNA-damaging agents are frequently used as chemotherapeutic drugs because cancer cells are vulnerable to DNA damage due to their increased genomic instability. As the replication fork encounters DNA damage the fork will stall. DNA repair after fork stalling can be studied in cells by treatment with hydroxyurea (HU), which depletes the nucleotide pools within the cells. In response to fork stalling, single stranded DNA (ssDNA) is exposed. The ssDNA is a target for the DNA repair proteins RPA, BRCA2, and RAD51. BRCA2 and RAD51 trigger repair of the stalled fork by homologous recombination (HR). Cells that are deficient in HR develop genomic instability, which drives tumor progression. HR repair is regulated by sumoylation, which is the addition of a small peptide (SUMO) to target proteins. Attachment of SUMO is catalyzed by E3 SUMO ligases, such as MMS21. Cells depleted of MMS21 are hypersensitive to DNA damaging agents.
We have shown that loss of MMS21 results in HR inhibition and cells lacking MMS21 have a lower production of ssDNA and RPA. Interestingly, cells lacking MMS21 also show larger and brighter RAD51 foci in response to HU, suggesting cells may be accumulating toxic HR intermediates. To test whether cells can perform HR effectively, we analyzed SCEs and found a drastic decrease in the number of crossover events, indicating that HR is non-functional. We also tested a newly approved chemotherapeutic (Olaparib), which acts by exploiting HR defects (such as BRCA2 deficiency) and found that cells were hypersensitive after MMS21 was removed (data not shown). Cells lacking MMS21 do not produce the DSBs necessary for proper repair during fork stalling while responding to radiation induced damage normally.
MMS21 may regulate the stability of RAD51 on the ssDNA, promoting turnover of RAD51 at the repair site. This process is critical for the normal resolution of HR intermediates. Release of RAD51 could be triggered by the activity of nucleases or fork remodeling proteins which promote the production of DSBs after long term fork stalling. These targets may be activated by MMS21 and “license” the repair of the fork. Understanding the regulation of DNA repair in cancer is essential for evaluation and effective use of DNA repair biomarkers. This project will benefit precision medical treatment through more accurate assessment of DNA repair deficiencies and continue to guide chemotherapeutic treatment decisions