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Environmental Factor, January 2012

Kleckner offers glimpses inside a living E. coli cell

By Jeffrey Stumpf

Nancy Kleckner, Ph.D.

LMG fellows invited guest lecturer Nancy Kleckner (Photo courtesy of Nancy Kleckner)

Richard Gradman, Ph.D.

Gradman is a member of the LMG Spontaneous Mutation and DNA Repair Group. (Photo courtesy of Steve McCaw)

During a Nov. 21 seminar at NIEHS, molecular biologist Nancy Kleckner, Ph.D., bypassed decades of her own groundbreaking research in the biology of prokaryotic transposons and meiosis, to focus on recent exciting work in the physical biology of chromosomes. Hosted by NIEHS postdoctoral fellow Richard Gradman, Ph.D., Kleckner spoke as part of the Laboratory of Molecular Genetics (LMG) Fellows Invited Lecture series.

Kleckner, (http://www.hms.harvard.edu/dms/virology/fac/Kleckner.php)  the Herchel Smith Professor of Molecular Biology at Harvard University, entertained the NIEHS audience with stunning images of dynamically changing chromosomes in cells of the model bacteria E. coli. While DNA research mostly focuses on molecules that affect basic processes in maintenance, replication, and repair, Kleckner provided a different angle with research describing how physical and mechanical forces might underlie chromosome behavior and function.

Stress and stress relief could explain the even spacing of meiotic crossovers

Correctly assuming an audience of physics novices, Kleckner provided the following example. Pulling on a rubber band will cause it to come under tension to produce what is called tensile stress. Cutting the rubber band will alleviate that stress not only at the point of the cut, but also along the length of the rubber band, a phenomenon termed redistribution of stress. The same principle will apply analogously to imposition and relief of pushing or compression stress. This principle, Kleckner argues, could underlie the even distribution of crossover events that occur during meiosis.

“All mechanical systems include such redistribution of stress, which in effect provides a method of communication,” Kleckner remarked. “Even spacing of the crossover events requires communication along the chromosome, because the positions of these events are not genetically specified. Instead, occurrence of an event at one position intrinsically disfavors the occurrence of another event nearby.”

If the site of a crossover is determined by a stress-promoted process, the result will be reduction of stress locally, resulting in reduced probability of another nearby crossover.

Crossover events during meiosis are required for segregation of meiotic chromosomes. Defects in crossing over, or their distribution along chromosomes, can lead to nondisjunction, the improper separation of chromosomes to gametes. Thus, considerations of how crossover events are located may be important in studying nondisjunction in humans. Kleckner mentioned that the nondisjunction rate in human cells can be up to 10 percent, but admits that studying the many causes for nondisjunction represents what she described as a whole other line of work.

The tangled web that cells weave

Questioning the possibility of an organized shape of the DNA of bacterial chromosomes, called nucleoids, Kleckner showed images of nucleoids captured by epifluoresence microscopy. Time lapsed images demonstrated that DNA in E. coli maintains a dynamic shape described as a helical ellipsoid, which can be either left-handed or right-handed. Kleckner showed videos that showcased how the nucleoids, which have a substructure comprised of dynamic longitudinal bundles, move elegantly as definable objects. “The DNA has a shape,” she joked. “It’s not just a bag of spaghetti.”

So what determines the nucleoid shape in E. coli? Kleckner explained part of the answer by means of a concept called radial confinement. Her observations suggest that bacterial cells use shape to separate their chromosomes, as a primordial precursor to the more complicated filamentous network of the mitotic spindle found in eukaryotic cells.

“The nucleoid is an ellipsoid that is longitudinally very stiff or, in technical terms, the persistence length of the ellipsoid is greater than the radius of the cell cylinder,” Kleckner observed. “During DNA replication, this ellipsoid evolves into a helicoidal shape, because it is forced around the cell periphery and held in place by friction, like a rubber rod trapped in a cylinder. As the two sister nucleoids emerge during replication, the stress resulting from this confinement increases until, finally, the two entities force their way into an end-to-end relationship.”

(Jeffrey Stumpf, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group.)




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