Environmental Factor, December 2009, National Institute of Environmental Health Sciences
Cellular Effects of Mutated DNA Polymerases
By Robin Arnette
DNA polymerase is responsible for DNA replication, a process that allows all living organisms to precisely copy their genetic material so that the information can be passed on to the next generation. But what happens when the polymerase has a mutation in its sequence? Does this genetic mistake produce abnormalities during replication and cause further harm to cells?
According to Stanford University School of Medicine's Teresa Shu-Fong Wang, Ph.D., cell lines that express a mutated polymerase experience chromosome chaos. Wang presented an NIEHS Distinguished Lecture on October 26 titled "The Perils of Bad DNA Polymerases: Chromosome Chaos." Bill Copeland, Ph.D., a principal investigator in the Laboratory of Molecular Genetics and a fellow expert in DNA replication, hosted the seminar.
Biochemistry and yeast genetics were Wang's specialty for many years, so she applied that expertise to human genetics and cell biology in her latest research. Wang explained the impetus for her work by saying, "I wanted to know what kind of chromosome chaos a bad or mutant DNA polymerase would cause, besides making genetic mistakes in the genome."
To address the question, Wang generated cell lines expressing either a mutated DNA polymerase α (Polα), the principal initiation polymerase, or a mutated DNA polymerase delta (Poldelta), a key elongation polymerase in normal human fibroblasts, cancerous p53-deficient HeLa cells and p53-proficient U2OS cells. Both mutations in these polymerases were in the evolutionarily conserved N-terminal region - not in the catalytic domain.
Wang said in normal human fibroblasts, expression of either polymerase compromised the S-phase progression in the cells. Polα-mutant expressing cells, in response to S-phase progression delay, were unable to maintain mitotic arrest with weak expression of mitotic checkpoint proteins BubR1 and Mad2. As a result, the cells experienced mitotic slippage and arrested in G1 as tetraploids, having four sets of chromosomes.
In contrast, the Poldelta-mutant expressing cells were able to maintain mitotic arrest in response to S-phase progression delay with proficient levels of BubR1 and Mad2. These cells had a normal number of chromosomes known as ploidy. Wang added, "These studies suggested that the mitotic checkpoint played a key role in preventing mitotic slippage that could result in polyploidy in replication-stressed cells."
In Polα-mutant expressing HeLa or U2OS cells, the Polα-mutant protein was unable to interact with And-1, a sister chromatid cohesion related factor. The Polα-mutant expressing cells exhibited mitotic and cohesion defects, resulting in chromosome chaos. The Poldelta-mutant expressing HeLa or U2OS cells induced dramatic deregulation of centrosome duplication, resulting in chromosome chaos. The cause of the centrosome duplication deregulation was due to the upregulation of cyclines E and A - proteins that control cell cycle progression - when cells experienced replication stress.
Wang concluded her talk with an important take home message. "In normal human cells, replication delay requires mitotic checkpoint to prevent mitotic slippage that will result in polyploidy of chromosomes. In cancerous cells, proper assembly of an initiation complex is a pre-requisite of the sister chromatid cohesion establishment, failure of which will result in chromosome chaos," she urged. "Defects in the replication elongation process will upregulate cyclins E and A to push the cells to go through S-phase, resulting in deregulation of centrosome duplication."
Wang used cell biology techniques to demonstrate that mutations in replicative DNA polymerases outside of the catalytic domain can cause a variety of chromosome abnormalities. Copeland, who completed his postdoctoral fellowship with Wang at Stanford, was familiar with her forward-thinking approach to science. He added, "Dr. Wang's ability to apply her previous experiences as a biochemist and yeast geneticist in cell biology demonstrates that she is not hesitant to take on new challenges."