Environmental Factor, September 2011, National Institute of Environmental Health Sciences
Fuchs discusses replication past DNA damage
By Jeffrey Stumpf
In the course of his presentation, Fuchs also invited postdocs to visit beautiful Marseille, France and study DNA replication past damaged bases in his lab at the Centre national de la recherché scientifique. (Photo courtesy of Steve McCaw)
In addition to hosting Fuchs's visit, Sarah Swerdlow, Ph.D., moderates a stimulating question and answer session after Fuchs's presentation. Swerdlow is a postdoctoral trainee in the NIEHS Mechanisms of Mutation Group, which also studies mutagenesis due to changes in nucleotide pools. (Photo courtesy of Steve McCaw)
Schaaper, right, and Mark Itsko, Ph.D., left, ponder the conclusions from Fuchs' studies. Schaaper leads the Mechanisms of Mutation Group in which Itsko performs his postdoctoral studies on E. coli genetics. (Photo courtesy of Steve McCaw)
Michael Resnick, Ph.D., right, takes note of Fuchs' findings during the lecture. Resnick is the head of the Chromosome Stability Group at NIEHS. (Photo courtesy of Steve McCaw)
Robert Fuchs, Ph.D.(http://www.cnrs.fr/index.php) , reacquainted himself with many familiar faces at NIEHS and shared his research about mechanisms of replication past damaged DNA, during his talk Aug. 1 at NIEHS. The renowned DNA polymerase expert from the Centre national de la recherché scientifique (CNRS) in France presented his discoveries for the Laboratory of Molecular Genetics (LMG) Fellows Invited Guest Lecturer Seminar Series.
Environmental factors and carcinogens cause DNA-damaging lesions that, if not removed, block replication by DNA polymerases. The replication machinery must bypass the damage, in a process called translesion synthesis (TLS), or abort replication. Because abortive replication leads to genome instability and cell death, TLS is advantageous for cell survival, but is mutagenic.
Fuchs uses the bacterium E. coli to study genetic controls that maintain DNA damage tolerance. NIEHS LMG researcher Roel Schaaper, Ph.D., also studies E. coli DNA replication and believes that the contribution of multiple polymerases to mutagenesis is important for environmental health.
“The sustained work by Robert Fuchs over the last two decades has been instrumental in teaching us about the roles of the various DNA polymerases,” Schaaper explains, “particularly when DNA replication encounters a blocking DNA adduct induced by environmental chemicals or ultraviolet light.”
Using plasmids to measure mutagenic TLS
Fuchs pioneered a plasmid-based system that studies the genetic mechanisms for bypassing replication-blocking lesions. The plasmid, a small circular strand of DNA, contains four important attributes:
- An antibiotic resistant gene, to select for cells with plasmids
- A damaged base
- A short region of unique sequence on the strand with the damaged base to identify plasmids that resulted from TLS
- A mutant variant of lacZ near the lesion to measure mutagenic TLS as lacZ mutations that allow wildtype function to make the colony turn blue
Although plasmids are convenient for genetic engineering, Fuchs is also ready to expand his studies to other replication mechanisms. “In the plasmid system, we can completely measure mutagenic and error-free TLS but not the so-called damage avoidance pathways, and now we are working on a system that will allow us to measure damage avoidance and TLS at the chromosomal level,” Fuchs explained.
Increased dNTP concentration pushes polymerases past the lesion
A long-standing observation from Fuchs' research is that TLS occurs at a low frequency under normal conditions, but 20-fold more frequently after UV exposure. The increase of the levels of DNA polymerase V (Pol V), which is necessary for replicating past the UV-damaged base, is presumably responsible for the increase of TLS. However, when Pol V was artificially induced without UV exposure, TLS frequency remained low. So, what else was necessary for UV-induced TLS?
To answer this riddle, Fuchs reported that UV exposure increases ribonucleotide reductase, the protein that limits the production of the building blocks of DNA or deoxyribonucleotides (dNTPs), and therefore increases dNTP concentration. Strains that increase both dNTP and Pol V concentration in the absence of UV allowed TLS. UV exposure only increases dNTPs 3-fold, but was enough, Fuchs argues, to tip the delicate balance for TLS.
“The level of dNTP is normally low, so we are working in a range where small changes matter,” postulated Fuchs.
Small changes in dNTPs matter especially for DNA polymerases. The genome is mostly replicated by DNA polymerase III complexes (Pol III), which contain an exonuclease protein that proofreads mistakes if the wrong nucleotide is incorporated. However, increased dNTP concentration favors replication rather than proofreading, and affects the interplay among DNA polymerases and exonucleases during TLS.
“What I find fascinating is to understand the genetic control of the response to increasing dNTPs,” Fuchs stated. “It's surprising that this hasn't been explored.”
Schaaper agrees, “The discovery that elevation of the dNTP levels is part of the DNA damage response, which improves survival at the cost of high mutagenesis, is an important new finding.”
(Jeffrey Stumpf, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group.)