DNA breakage surprisingly rare

DNA breakage during replication in Escherichia coli occurs at a frequency 20-100 times lower than previously thought, according to a report published this week in Nature Genetics, which describes a new technique to visualize strand breaks in vivo."We can be pretty confident...the researchers are as close as you can get [to the real number of breaks]," Aidan Doherty, at the University of Sussex Center for Genome Damage and Stability, who was not involved in the study, told The Scientist. Susan Rosenberg's lab devised a novel assay detection system and genetically programmed wild-type E. coli cells to glow green in the presence of a DNA double-strand break (DSB). This technique provided the first direct data on the number of DSBs actually occurring in prokaryotic cells. Previous research has estimated DSBs occurred in every chromosome generation. Rosenberg and her colleague, however, place the rate at approximately one DSB in every 100 chromosome replications.The lower frequency of breaks suggests individual breaks may each play a more severe role in causing damage to cells, mutation, and cancer.But even considering the newer, reduced rate, DSBs are surprisingly common, said Rosenberg. "Homologous recombination is the repair mechanism favored in E. coli, it's the most benign way the cell can put the DNA back together. It's supposed to be the error-free pathway," Rosenberg told The Scientist. A DSB occurring every one in 100 replications equates to a 10% frequency of rearrangement, she added. "That's huge for what was supposed to be the error-free pathway."In order to directly observe the number of DSBs in the bacterial cells, Rosenberg and her colleague, Jeanine Pennington, both based at Baylor College of Medicine, inserted a gene encoding the green fluorescent protein into a spot in the bacterial genome used by viruses to infiltrate the host. This avoided the chance that manipulation of the genome would cause experimentally-induced results by influencing the expression of other genes, and insured that researchers would be observing practically wild-type cells. When a break occurs in the DNA during replication, the cell activates a set of mechanisms that rush to repair the damaged site. By Rosenberg's design, those mechanisms, when activated, caused the cells to fluoresce.Using flow cytometry, the researchers literally counted the number of DSBs by counting the number of green cells in the assay. The researchers also performed a number of control experiments to eliminate the possibility that any other factors were causing the cells to turn green. For example, when the scientists eliminated the gene that helps activate the repair mechanisms, no cells turned green, helping to confirm that the fluorescence was triggered by DNA repair mechanisms only."Without these controls you'd be scratching your head saying 'is that real or not?'" noted Doherty.Previous estimates of the number of DSBs came from cruder, indirect tools, which estimated the breakage rate by counting dead cells in a culture, or smashing up the cell and then running a gel electrophoresis."It's an interesting study, mainly from the technology point of view," Errol Friedberg, at the UT Southwestern Medical School, who did not participate in the study, told The Scientist. "They have devised an elegant assay, and even though the work was done in E. coli, it should be 'extrapolate-able' to the eukaryotic system."The next step is to see if researchers can apply the same methods to higher-level cells. The number of genes that control breakage repair is greater in eukaryotic cells, creating a vast array of mechanisms associated with breaks, but the basic method of incorporating the green fluorescent protein should work, Rosenberg said. Since DNA breaks occur less frequently than believed, the eukaryotic cell is probably investing tremendous energy into maintaining the integrity of the DNA, suggested Doherty. "It's probably why, in eukaryotes, there has evolved a more complex mechanism of histones. They need to chaperone the DNA closely and not let it break."Andrea Gawrylewski mail@the-scientist.comLinks within this article:J. Pennington and S. Rosenberg, "Spontaneous DNA breakage in single living Escerichia coli cells," Nature Genetics, published online May 27, 2007 http://www.nature.com/ng/index.htmlAidan Doherty http://www.sussex.ac.uk/gdsc/1-4-5.htmlSusan Rosenberg http://imgen.bcm.tmc.edu/molgen/facultyaz/rosenberg.htmlM. Kastan, "DNA damage responses: Cancer and beyond," The Scientist, October 10, 2005. 'http://www.the-scientist.com/article/display/15766M.L. Phillips, "Commensal bacteria damage host DNA," The Scientist, August 10, 2006. http://www.the-scientist.com/news/display/24285/Errol Friedberg http://www.utsouthwestern.edu/findfac/professional/0,2356,12367,00.html

DNA breakage surprisingly rare

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