09 May UPMC Hillman Cancer Center Scientists Study How Telomere DNA is Damaged and Modified
MedicalResearch.com Interview with:
Dr. O’Sullivan
Roderick J. O’Sullivan PhD
Associate Professor
Department of Pharmacology and Chemical Biology
UPMC Hillman Cancer Center
University of Pittsburgh
Pittsburgh, PA
MedicalResearch.com: What is the background for this study?
Response: For a few years, my group has had the good fortune of collaborating with Dr. Ivan Ahel. Ivan is a world leader in the field of ADP-ribosylation. His work has identified major gaps in our understanding of ADP-ribosylation. This includes his lab discovering that DNA bases can be ADP-ribosylated in bacteria and that a poorly characterized enzyme known as TARG1 could be involved in that process. In discussing this work with Ivan, we were confident that DNA ADP-ribosylation also exists in human cells and that showing this could be pretty important. The problem was that identifying a part of the genome where it might be present, so we could study it, was not so obvious and challenging. But we had a hunch that telomeres could be one part of the genome where it could happen!!
Telomeres are really special structures located at the ends of each human chromosome. They demarcate the physical end of each chromosome and prevent chromosomes from becoming entangled – which if it happens, is catastrophic for cells. Our hunch was based on the evidence from other studies that telomeres are natural targets of PARP1, the enzyme that catalyzes most of the ADP-ribosylation in human cells. I then discussed this idea with Anne Wondisford, a medical scientist trainee in the lab, who liked the idea and designed a series of experiments to test it.
MedicalResearch.com: What are the main findings?
Response: Anne used a specialized method to isolate telomeric DNA from cells. She then used specific reagents that detect all-forms of ADP-ribosylation to examine if this modification is present on the DNA that she isolated. The trick here was that Anne had first used CRISPR to delete TARG1, the enzyme which Ivan suspected is essential for degrading DNA-linked ADP-ribosylation. By deleting TARG1, the idea was that the DNA-ADP-ribosylation cannot be removed and should then give us a better chance of detecting it. Sure enough – when Anne did a basic western blot for ADP-ribosylation, the samples from TARG1 knockout cells lit up positively. This was the first result that indicated we were onto something potentially new and interesting.
In speaking with Ivan and his postdoc Marion Schuller, we decided to test whether taking the telomere DNA and incubating recombinant TARG1 enzyme would remove the ADP-ribose. Indeed, this is what happened, confirming that TARG1 is the key enzyme to remove ADP-ribose from DNA. We then decided to verify if PARP1 is the enzyme that adds ADP-ribose to DNA.
Here we used Olaparib, a cancer therapeutic that inhibits PARP1 activity to treat patients with BRCA mutated breast and ovarian cancer. When we added Olaparib to cells and performed our analysis, we found that the DNA-ADPr of telomeric DNA completely vanished. This pretty conclusively showed us that telomere DNA is modified by PARP1, and the ADP-ribose is removed by TARG1.
With this knowledge, we could then ask some very basic but fundamental questions like; when is telomeric DNA modified and What does it do? From this first analysis, Anne found that telomere DNA is modified following DNA damage and importantly during S-phase – the crucial time when DNA strands are replicated. She found that DNA-ADP-ribosylation is very intimately associated with telomere replication and the failure to remove ADPr damages telomere structure and causes premature loss of telomeric DNA which is very bad for the stability of the genome.
MedicalResearch.com: What should readers take away from your report?
Response: I think an important ‘meta’ message is how we are still learning some pretty fundamental things about ADP-ribosylation, even though we’ve been studying it for more than 50 years. It has become clearer that PARP inhibitors affect DNA ADP-ribosylation. This could be an important consideration for future PARP inhibitor development and testing.
I also suspect that this is the tip of the iceberg. We have no idea of where else in the genome DNA is ADP-ribosylated, but it is likely to be more common than we suspected. We also have a lot to learn about whether DNA-ADP-ribosylation has any functions in particular cancer subtypes or in specific tissues. We are gonna learn a lot more about this new type of ADP-ribosylation over the next few years.
MedicalResearch.com: What recommendations do you have for future research as a results of this study?
Response: As alluded to before, it is very likely interfering with PARP in cancer, through PARP inhibitors, affects DNA-ADP-ribosylation. Whether defects in DNA-ADPr contribute to the mechanism of cancer cell death is unknown. But it is likely to be so. I think understanding the regulation of DNA-ADPr in cancer cells, including BRCA mutated cancer subtypes, could be important.
MedicalResearch.com: Is there anything else you would like to add? Any disclosures?
Response: Just that this study was born out of many zoom calls during the pandemic with great colleagues. We could not have done this work without Ivan Ahel’s team in Oxford and Hilda Pickett’s team in Sydney. We were also very lucky to get advice and help from Patty Opresko here in Pittsburgh and Jaewon Min at Columbia University. Collaborative science is a lot of fun!!
No disclosures
Citation: Wondisford, A.R., Lee, J., Lu, R. et al. Deregulated DNA ADP-ribosylation impairs telomere replication. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01279-6
https://www.nature.com/articles/s41594-024-01279-6#citeas
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Last Updated on May 9, 2024 by Marie Benz MD FAAD