LinE proteins pave the way for determining DNA breakage hotspots


Like two boxers returning to their corners at the end of a round, it is imperative that duplicate chromosomes separate on opposite sides of a cell during cell division. But in this process lurks a striking paradox – to faithfully separate, these chromosomes must first be glued together. This bonding provides tension as the chromosomes begin to move toward their respective corners, and “the tension signals that the centromeres are going to opposite poles of the cell,” describes Dr. Gerry Smith, a professor in the Fred Hutch Division of Basic Sciences. and at UW/Fred Hutch Cancer Consortium Member. During mitosis, the chromosomes are glued together like glue. During meiosis, the connection is much more intimate – the two chromosomes are separated and then joined together by a process called crossing over which results in their direct physical coupling. In a new research article in the journal Nucleic acid researchDr. Smith’s group examined how these cross-events are regulated in yeast cells.

Crossover begins with the formation of DNA double-strand breaks (DSBs), which can then be repaired to restore normal chromosome structure or recombined to generate crossovers. “DSBs are not evenly distributed across chromosomes. Rather, there are sites, called DSB hotspots, at which DSBs occur preferentially,” Dr. Smith’s group wrote. A group of proteins called LinE proteins – namely Rec25, Rec27 and Mug20 – localize to hotspots and are required for the formation of DSBs there. However, a lack of understanding of how LinE proteins are recruited to hotspots has “precluded testing whether LinE protein binding to a chromosomal site is sufficient to create a DSB hotspot,” the authors wrote. To overcome this limitation, they devised a clever new strategy to promote the binding of LinE proteins to a site of their choice. They introduce a specific DNA sequence, called lacOin a region of the fission yeast genome devoid of hotspot activity and then fused the LinE proteins to a protein of E.coliLacI, which binds the lacO sequence. The resulting forced localization of LinE proteins caused large increases in DSB formation and up to a ten-fold increase in recombination at the lacO to place. In addition, the recombination at lacO The site used the same molecular machinery as in endogenous hotspots, indicating that LinE protein recruitment is sufficient to create a DSB hotspot. The authors, however, noted a discrepancy between the frequencies of DSB formation and recombination at this site – they observed much less recombination than expected for the number of DSBs generated at this site, suggesting that many DSBs were resolved without generating of crossings. This discovery also indicated that their artificial hotspot may not have been a perfect replica of an endogenous hotspot.

the lacO/The LacI strategy further allowed the authors to examine another intriguing but poorly understood feature of DSB hotspots. “DSB hotspots do not act independently – they interact with neighboring hotspots,” the authors explained. These interactions include DSB competition, in which the introduction of a new hotspot reduces the frequency of DSB formation at nearby hotspots, and DSB interference, in which the formation of a DSB at a hotspot decreases the probability of formation of a second DSB at a nearby hot spot. . “In cells with wild-type LinEs, DSBs show both competition and interference,” the authors noted. However, in a result they called “remarkable”, the group found that the lacO the hotspot presented almost no competition with nearby endogenous hotspots; in fact, a nearby access point has become more active. Conversely, the lacO hotspot displayed DSB interference with a nearby hotspot, highlighting the separability of these two forms of hotspot interactions.

Dr. Smith wanted to emphasize the level of teamwork involved in this project. “This work started with Josh Cho, a technician, about 8 years ago. He has carried out and ambitiously tested numerous fusions of the LacI protein with each of the proteins of the linear elements and has carried out several lacO insertions in a gene (ade6) well suited for measuring recombination and DNA break formation. He found a range of activities and showed that they worked as expected. Unexpectedly, postdoc Mridula Nambiar discovered that there were 10 times more DNA breaks than expected from recombination frequencies. Research scientist Randy Hyppa then showed, through extensive work using techniques he developed, that the high:low recombination ratio is due to higher than expected repair of DNA breaks with sister, which cannot give genetic recombinants. And it showed that the DNA break hotspot created by Josh had DNA break interference but no competition. Following this, Hyppa explained that he was particularly excited by the band’s observation that the lacO hotspot has no DSB concurrency. “I think the most important contribution is that the data can help us reveal which factors are important in establishing competition for DNA double-strand break (DSB) hotspots. It has long been known that the introduction of a strong DSB access point will remove existing nearby access points, but it is still unclear how it works. It seems that rows that aren’t loaded by the usual mechanism result in DSB APs that don’t show concurrency. This insight will allow us to target the protein complexes involved in DSB competition, which we currently believe to be cohesin and condensin DNA complexes. We have ongoing experiments to examine DSB competition in cohesin mutants, and we are also examining how the cohesin complex interacts with LinE proteins.


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