A new study shows that despite inhibition of the essential gene DNA polymerase alpha, POLA1, cells can survive and multiple, according to research from the Center for Chromosome Stability at the Department of Cellular and Molecular Medicine, University of Copenhagen. However, there is a limit to that capacity, and this new insight could lead to new anti-cancer drugs— POLA1 inhibitors— the researchers suggest.
The studies were led by associate professor Luis Toledo and were published recently in Cell Reports, “Physiological Tolerance to ssDNA Enables Strand Uncoupling during DNA Replication.”
It has been long assumed that leading strand synthesis must proceed coordinated with the lagging strand to prevent strand uncoupling and the accumulation of single-stranded DNA (ssDNA) in the cell, which is eventually toxic. But these researchers found that human DNA polymerases can function independently at each strand in vivo and that the cell can withstand resulting strand uncoupling.
When a cell divides, the double DNA strand is opened lengthwise like a zipper that is unzipped. The new double strands are built at each of the separated strands, so two new “zippers” are created, according to the group’s press release. Before the new halves of the zipper are made, a bit of DNA is temporally exposed in single stranded form. This process is required for the new zippers to form: Large amounts of single-stranded DNA have traditionally been considered to be a sign of pathological stress during cell proliferation.
However, the University of Copenhagen researchers find that DNA unzippers act more loosely than expected. This can generate large amounts of single-stranded DNA, which the researchers find is no more than a form of natural stress that cells can actually tolerate in high quantities. Still, for this tolerance to exist, cells require a sufficient amount of Replication Protein A (RPA) to cover the single-stranded DNA parts.
“We have seen that cells can duplicate their genome, even with large amounts of single- stranded DNA. They can divide and go on living healthily because they have a large excess of RPA molecules that acts as a protective umbrella,” says the study’s first author and former postdoc at the University of Copenhagen, Amaia Ercilla, adding: “But there is a flip side of the coin. When we make the cells generate single strand DNA faster than what they can protect, chromosomes literally shatter in hundreds of pieces, a phenomenon we call replication catastrophe. We always thought that we could use this for instance to kill cancer cells.”
Under normal circumstances it is extremely difficult to deplete a cell’s reserve of RPA. The same was true in the new study, when researchers used different types of chemotherapy to increase the amount of single-stranded DNA. Even when using the best compounds available so far it took around one hour to deplete the RPA reserve in a cell, provoking a replication catastrophe and the associated cell death.
However, the researchers believe they have found what Toledo calls “the ultimate single-stranded DNA generator.” When the researchers used a POLA1 inhibitor, the cells died after just five minutes.
“Although no new DNA can be made when we inhibit POLA1, the DNA unzippers keep advancing and generate single-stranded DNA at very high speed,” says Toledo, adding:
“All cells can be sensitive to POLA1 inhibitors, including cancer cells, and we might speculate that the strategy could be especially useful against very aggressive forms of cancer that proliferate at a high pace.”
The next step of the research group is to find more molecules that biologically inhibit the POLA1 gene and which, in combination with other substances, may be used in the treatment of cancer patients.