Taking a closer look at DNA double-strand break hotspots

From the Smith Lab, Basic Sciences Division

Accuracy of observation is the equivalent of accuracy of thinking. -Wallace Stevens

Experimentation may be what first comes to mind when one contemplates the process of scientific research, but sometimes the best way to understand a system is to simply observe it in its native state. Thus, a great component of innovation in biology has always been to improve our ability to see structures and events within living systems. Microscopy has been a force in the field since its invention in the 17th century. And in recent decades, fluorescence microscopy, which allows for high-contrast visualization of structures within the cell, has again transformed what we are capable of seeing. But even with the most powerful of light microscopes, things are not always what they appear. Some biological structures are simply too small to be resolved, leading them to appear as mere blobs despite what intricate forms they may truly possess. Recently, scientists have developed optical techniques to circumvent these limitations, spawning the field of super-resolution microscopy (awarded the 2014 Nobel Prize in Chemistry) which has begun to allow us to observe the true structures underlying what were once only blobs. In a new article published in Journal of Cell Science, Dr. Gerry Smith, a professor in Fred Hutch’s Basic Sciences Division, and research fellow Dr. Yu-Chien Chuang use this powerful technique to observe DNA double-strand break hotspot protein complexes and discover a previously unappreciated variability in their structures.

The regulated formation of DNA double-strand breaks (DSBs) is a key step in meiosis. In fission yeast, a group of proteins called the Linear Element (LinE) proteins localize at sites in the genome known as DSB hotspots to promote DSB formation. Previous attempts to visualize the structure of the LinE complex at hotspots in fixed cells had been plagued by conflicting results – sometimes they appeared as dots, sometimes as more elongated linear structures, depending on the fixation conditions and the temperature at which the cells had been grown. Which of these views, the authors wondered, truly reflected the state of LinE in a living cell? To answer this question, they grew cells in conditions that most closely matched their wild-type state and used super-resolution microscopy to visualize fluorescently-tagged LinE proteins on the DNA of live cells.

While the general view in the field was that LinE tends to be more dotty, the authors readily observed long linear structures in meiotic cells. Examination across the cell cycle revealed LinE to be surprisingly dynamic – early in meiosis, LinEs were mostly dotty, followed by a transition to mostly linear structures mid-meiosis and then back to dots towards the end of meiosis, in a process the authors termed a “dotty-to-linear-to-dotty transition”. Thus, it seems that the best answer to whether LinE forms dotty or linear structures is, in fact, “both”. This finding alters how we should consider LinE, says Dr. Chuang. “The fission yeast S. pombe is thought by many to lack the meiotic synaptonemal complex (SC) present in most other organisms,” she explained. But the dynamic structural transitions she observed are quite similar to the SC, suggesting fission yeast may not be so distinct from other organisms as previously believed.

LinE timecourse
Mitotic time-course of LinE structures (green) on DNA (blue) in the nuclei of live cells (dashed outline) showing the dotty-to-linear-to-dotty transition at the indicated time after initiation of meiosis. Image provided by Dr. Yu-Chien Chuang.

After clarifying the structural details of the LinE complex in wild-type cells, the authors used their system to examine the impact of experimental perturbations on this structure. First, the authors found that removing any one LinE component protein generally affects the ability of the others to form nuclear structures, suggesting they work closely together in this task. They next blocked the generation of DSBs and found that this delayed the disassembly of LinE structures, suggesting their dynamics are tied to DSB formation. Finally, the authors introduced mutant versions of LinE proteins that are deficient in promoting DSBs and DNA recombination and found that these were deficient in forming nuclear structures and in undergoing dotty-to-linear-to-dotty transitions, indicating these structural dynamics may be important for LinE function.

Moving forward, Dr. Chuang is keen to understand the functional relevance of LinE structures. “We are especially interested in the dynamics of the LinE complex – how the two configurations relate to DSB formation and repair, and perhaps to DSB distribution and crossover interference…The two configurations of the LinE complex also suggest it has multiple functions in meiosis. We further showed some LinE recombination-deficient mutants form dotty but not linear structures, indicating the linear LinE may regulate DSB repair,” she explained.  “Live cell imaging is a relatively new method in our lab,” she continued, though one with great potential to continue to unlock the mysteries of this tiny protein complex.

This work was supported by the National Institutes of Health.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Gerry Smith contributed to this work

Chuang YC, Smith GR. Dynamic configurations of meiotic DNA-break hotspot determinant proteins. J Cell Sci. 2022 Feb 1;135(3):jcs259061. doi: 10.1242/jcs.259061. Epub 2022 Feb 7. PMID: 35028663; PMCID: PMC8918816.