This study was published in Thu et al., Cell Reports, 2016.
One essential process of life, known as DNA replication, is to accurately pass on the inheritable genetic information to the future generation. Intriguingly, replication is an orchestrated process involving multiple molecular players. Disrupting the function of any of these players result in a form of cellular stress known as replication stress. This condition, if remained unresolved, can introduce errors in the genome and therefore be deleterious to cells. Replication stress is in fact a forerunner of cancer. Fortunately, cells evolve multiple cellular mechanisms that are set in motion to counter-balance the compromise. My post-doctoral research effort was to understand some of these mechanisms.
We used a mutant of Mcm10, a replication protein which is essential for cell survival. A mutation that we used in MCM10 gene compromised cells’ ability to accurately copy the genome, leading to replication stress. We were interested in genes that were specifically necessary when MCM10 was absent. In other words, we were on the hunt for genes required to protect cells from replication stress. From this effort, we identified SLX5 and SLX8. When SLX5 or SLX8 gene was absent, cells with the MCM10 mutation grew poorly. Intrigued by the data, we wanted to find out how Slx5 and Slx8 proteins might protect cells from replication stress.
We had some clues from previous studies which have looked into these proteins’ function. It turns out that Slx5 and Slx8 work together as a protein complex. They are enzymes that mark other proteins for degradation. Protein degradation is one strategy cells use to terminate a biological process. If the participant proteins are destroyed, the biological process will grind to a halt. Therefore, we ask the question: what biological process or pathway is being turned off by Slx5/Slx8 complex under stress?
This question was answered by isolating all potential target proteins that Slx5/Slx8 may mark for degradation and identifying them what they were by proteomics. We found approximately a hundred proteins that may be targeted by Slx5/Slx8 in cells experiencing replication stress. Which ones did we think were involved in a process, when interrupted, would rescue cells from replication stress-induced death?
We used bioinformatics tool and a large body of knowledge from the literature to answer this question. We decided to hone in on a group of proteins involved in checkpoint signaling. This makes sense because checkpoint signaling pathways are surveillance forces of a cell. If, for example, the genome is damaged, checkpoint pathways stop cells from growing or dividing until the problem has been resolved. In other words, checkpoint mechanisms prevent propagation of faulty materials. However, this genome integrity check comes at a price. Cells that cannot escape the checkpoint due to persistent DNA damage will eventually die. We reasoned that checkpoint proteins were perhaps degraded by Slx5/Slx8, relieving the break which would otherwise stop the cell cycle. Our data from both genetics and biochemical experiments support this model.
In essence, we demonstrated the existence of a pathway that provides another chance for cells to repair the damaged genome. When cells are faced with the burden of genome instability, checkpoint signaling may prevail. Alternatively, replication stress resistance mechanisms such as one mediated by Slx5/Slx8 may ensue, allowing cells to repair the damage during the next round of cell cycle.
Intriguingly, the balance between replication stress tolerance and checkpoint-mediated cell cycle arrest/death is intricately linked to cancer biology. Due to uncontrolled proliferation, cancer cells are constantly hampered by replication stress. Yet, they somehow manage to be alive. Understanding the mechanism underlying replication stress resistance will give us clues on how to specifically target cancer cells.