2022 年度 第４回 生命科学技術国際卓越講義
World-leading Innovative Lecturesin Life Science & Technology
The University of Tokyo

Seeing is believing: break-induced mitotic recombinationfrom 1936 to 2022
Prof. Rodney Rothstein
Professor, Department of Genetics and Development,
Columbia University Medical Center

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Date: Thursday, 17th, November, 2022
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Time: 5：00PM ~ 5:50PM Lecture
6:00PM ~ 6:30PM Interview Session with Students and Young Researchers(Attending an interview session needs another registration (see your e-mail))

Venue: Zoom (meeting URL will be sent after registering)
Participants: Up to 500 participants

Abstract:In 1936, Curt Stern showed that “twin spots” arose in Drosophila after a mitotic
recombination event between the centromere and two distal markers that were linked inrepulsion (y, yellow body color and sn, singed bristles; Genetics 21: 625–730). His analysis
suggested that, after the crossover, one of the two chromosome segregations that can takeplace during mitosis leads to Loss of Heterozygosity (LOH). The other mitotic segregation
does not lead to LOH, although one daughter cell contains the two reciprocally crossedover chromosomes. Stern hypothesized that the crossover had to occur in G2 to generate
the LOH. To examine the steps in mitotic recombination that occur after induction of asite-specific double-strand break (DSB) in diploid budding yeast cells, we have created astrain that contains “Yellow” and “Red” fluorescent tags flanking each side of an I-SceI
cut site. The homolog, without a cut site, contains a single “Magenta” fluorescent tag at
the homologous position of the Red site. Crossover events can be visualized by the change
in linkage of the Yellow tag with respect to Red and Magenta. In addition to the I-SceI enzyme, we have designed a CRISPRCas9 guide that also directs cutting very close to the I-SceI site. Thus, we can compare the repair outcomes of an I-SceI-generated
break (3’-protruding end) to a CRISPR-Cas9 break (blunt end). In both cases, nuclease expression is induced continuously and>99% of the surviving colonies undergo gene conversion of the cut site. Cells from the colonies are examined using
epifluorescence microscopy to determine whether the chromosomes have undergone crossovers associated with the induced geneconversion event. After I-SceI and CRISPR-Cas9 induction, crossovers and non-crossovers each account for approximately halfof the repair outcomes. For I-SceI, 10 of the 26 crossovers give rise to LOH and for CRISPR-Cas9-induced breaks, 4 of the 14
crossovers are LOH. Importantly, the colonies containing non-LOH crossovers have cells with two reciprocal recombinant
chromosomes, allowing us for the first time to directly visualize the mitotic segregation class that Stern predicted 86 years ago.
Finally, no G1 crossovers were recovered since “pure” non-LOH colonies were not observed. Our results fit a model whereby theefficient repair of a DSB in mitotic diploid cells results in a G2 crossover half of the time.
At the same time, we have also been studying how homologous recombination after induction of a DSB is linked tochanges in chromosome movement. We find that end resection, the recruitment of recombination proteins, increased chromosome
mobility, the pairing of homologs and gene conversion are temporally linked in response to a DSB. In mre11∆ mutant cells, which
exhibit a delay in the initial processing of a DSB, chromosome mobility and all subsequent recombination events are also delayed.
Interestingly, overexpression of the Dna2 nuclease, but not ExoI, suppresses the mre11∆ delay in end resection and restores theoriginal timing of chromosome mobility and all subsequent downstream HR events. Thus, changing the timing of chromosome
mobility results in a corresponding change in essential downstream HR events, reinforcing its mechanistic role in the DNA repair
process.
Organizer: World-leading Innovative Graduate Study Program for Life Science and Technology
Cooperation: Graduate Program for Leaders in Life Innovation, The University of Tokyo
Clinical Research Promotion Center, The University of Tokyo Hospital
For Further Information Contact: Kotoko Ogawa at WINGS-LST Office Phone: 03-5841-3483 E-mail: kogawa@g.ecc.u-tokyo.ac.jp