Exposure to reactive oxygen species, exposure to ionizing radiation, exposure to UV light – all of these are dangerous because of their potential to alter DNA sequences. Changes in DNA can affect a protein coding sequence, potentially influencing its function, but changes in regulatory regions can be equally dangerous if they significantly change a promoter or ribosome-binding site.
No matter where they reside - obligate intracellular resident or thermophilic extremophile - all microbes experience some of these stress types. Each cell needs to counteract potentially mutagenic stresses to provide an undamaged genome to progeny cells. One of the most resilient bacterial species described, Deinococcus radiodurans, has an impressive ability to repair its DNA. The microbe was discovered when irradiated meat spoiled, and D. radiodurans was found to be the cause; its DNA repair abilities are thought to be one reason this bacterium can survive such harsh conditions. New research, published in ASM’s new mSphereTM journal, shows a link between DNA repair and chromosome segregation during cell division contributes to D. radiodurans robustness.
The research focuses on the Deinococcus-specific gene pprA, which codes for one of the DNA repair enzymes used by D. radiodurans. A pprA mutant is susceptible to gamma-irradiation and other DNA-damaging conditions, highlighting the role of this gene in resistance. In purified form, PprA protein binds preferentially to double-stranded DNA breaks, but inside the cell, PprA acts in concert with many other protective enzymes, including DNA gyrase, an enzyme important for unwinding the double helix during DNA replication and repair. The research team, headed by Dr. Pascale Servant, wanted to better understand PprA-DNA Gyrase interactions.
Using immunoprecipitation and mass spectrometry, the researchers showed that the two proteins interact after D. radiodurans cells are exposed to gamma irradiation. Suspecting that PprA interaction might influence the DNA gyrase function, the researchers showed that a ΔpprA mutant strain is hypersensitive to drugs that inhibit either of the subunits (GyrA or GyrB) that make up DNA Gyrase, demonstrating that PprA modulates DNA Gyrase activity.
The ΔpprA mutant strain also improperly segregates chromosomes during cell division, which the researchers thought might be related to PprA-Gyrase activity. Microscopic investigation showed a number of abnormalities in chromosomal segregation when the Gyrase inhibitor was added, with nearly 10% of cells not receiving a chromosomal copy (anucleated). The effect was intensified in the ΔpprA mutant, with nearly a third of the dividing cells being anucleated (see figure, right).
How could so many cells be improperly sharing their chromosomes during division? D. radiodurans has an anomalous number of topoisomerase proteins, the protein family that includes DNA Gyrase. In E. coli and other well-studied bacteria, an enzyme called Topoisomerase IV (Topo IV) is involved in separating chromosomes during cell division. D. radiodurans has no recognized Topo IV gene homolog, so the researchers hypothesized that DNA Gyrase fills this role.
They tested this hypothesis by incubating purified GyrA and GyrB proteins with catenated DNA; minicircles of DNA that are interlocked, similar to the way circular bacterial genomes are interlocked after replication (see example of interlocked plasmids, left). Topo IV normally untwists these by physically breaking and resealing the DNA backbone after untwisting it. Incubating purified DNA Gyrase with the catenated DNA decatenated (separated) the DNA, and this activity was further stimulated with the addition of purified PprA.
Taken together, this is evidence that PprA activity contributes to D. radiodurans chromosome maintenance after DNA damage from gamma irradiation, not only in DNA repair but also in chromosomal inheritance via DNA Gyrase stimulation. So one of the reasons PprA is so important to the cell – and one of the reasons a ΔpprA mutant strain is much more susceptible – is because of the multiple functions PprA performs toward protecting the cell’s DNA.
We are excited to see the new mSphereTM website launched soon; be sure to look on the website and check back at mBiosphere for more research highlights from the American Society for Microbiology!
-- Julie Wolf