Poster 107 - MRE11 Recruitment to DNA Double Strand Breaks After Laser-Ablation Inside U2OS Nucleus

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Michelle Cai

High School Volunteer Research Intern
University of California, San Diego Institute of Engineering in Medicine, California, United States

Co-Author(s)

SS

Sharanya Sridharan (she/her/hers)

High School Volunteer Research Intern
University of California, San Diego Institute of Engineering in Medicine
San Diego, California, United States

Co-Author(s)

Ryan Liu

High School Volunteer Research Intern
University of California, San Diego Institute of Engineering in Medicine
San Diego, California, United States

Co-Author(s)

Grace Xie (she/her/hers)

Undergraduate Student
University of California, San Diego
La Jolla, California, United States
LY

Luke Yang

Undergraduate Student
University of California, San Diego, United States
BH

Bryan Huang

High School Volunteer Research Intern
University of California, San Diego Institute of Engineering in Medicine, United States
Leo Meng

High School Volunteer Research Intern
University of California, San Diego Institute of Engineering in Medicine, United States
YL

Youhang Li

PhD Student
Scripps Research Institute, United States

Last Author(s)

XW

Xiaohua Wu

Professor
Scripps Research Institute, United States

Primary Investigator(s)

Linda Shi, PhD (she/her/hers)

OPALS Program Director, Project Scientist, Co-director IEM
University of California, San Diego: Institute of Engineering in Medicine
La Jolla, California, United States

Introduction: MRE11 is a significant protein-encoding gene that allows for DNA repair in cells. It encodes a nuclease involved in homologous recombination, telomere length maintenance, and most notably, DNA double strand break (DSB) repair. Mutations in MRE11 can have negative effects, including diminished repair efficiency, impaired activation of DNA checkpoints, and even total loss of function. Understanding the effect of specific mutations on MRE11’s ability to repair DSBs can provide insights into the molecular mechanisms behind diseases like cancer, in which DNA repair pathways in cells are often impaired. In order to more deeply understand the mechanisms of MRE11 participation in DNA DSB repair, both wild-type and different mutant cell lines were introduced to laser-induced DNA DSBs.

Materials and

Methods: Established protocols were followed for the culturing of U2OS cells[1], selected due to their widespread use and recognition as a standard model for cancer cell research. Standardized procedures were used to induce 2A, 2D and MT point mutations in the MRE11 genes which resulted in four distinct types of U2OS cell cultures with six cell culture dishes: two with Wild type MRE11, one with 2A MRE11, one with 2D MRE11 and two with MT MRE11. Images of the U2OS cells were taken approximately every 5 minutes for 30 minutes after ablation, exposing cells for one second at 50% power each time. Laser irradiation and imaging was performed on a tunable femtosecond mode locked Ti:Sapphire infrared laser coupled to Zeiss Axiovert 200m inverted microscope allowing targeting of subcellular regions[2]. The laser wavelength used in this project was 780 nm. Laser power was 50mW before entering the 100X 1.3 NA Zeiss phase contrast objective. The peak irradiance at the focal point was 1.06 X1012 W/cm2 for 780 nm. The ablation was applied along multiple dots by a scanning laser, occurring fast in sequence. The images collected from different time intervals were later imported to Fiji ImageJ where the protein aggregation was measured and recorded.

Results, Conclusions, and Discussions: Results and

Discussion: Using ImageJ, the intensity of protein aggregation was measured at each time interval. The resulting graph indicates that intensity increases as MRE11 gathers at the cut site, plateaus while the DNA DSBs are repaired, and decreases afterward. 2A, 2D, and WT MRE11 all had the greatest aggregation around 10 minutes after damage was induced, before gradually declining. However, MRE11 recruitment was higher on average in WT than any mutant type. The results of the 2D and 2A mutants indicate slight functionality in recruitment to DNA damage sites (higher in 2D than 2A), but recruitment is still significantly greater in WT. The MT mutant MRE11 had very little protein aggregation, and its intensity level stayed low from 0 to 30 minutes after damage was induced, indicating little to no recruitment to DNA DSB sites.

Conclusions and Future Study: Our data concludes that the effectiveness of proteins encoded by MRE11 is highly dependent on the state of the gene. As the results of this experiment have shown, mutations greatly decrease the ability of encoded proteins to make DNA repairs. These results are consistent with previous research and published findings on MRE11 recruitment effectiveness. Thus, the integrity of MRE11 genes may be a significant factor to investigate when preventing the progression of cancer or genome instability. Although our project was able to identify a decrease in DSB repair functionality associated with mutations, it is unclear as to exactly what changes such mutations make to the MRE11 protein that negatively affect repair activity. Future plans include optimizing the efficiency of image analysis by using CellPose to train a machine learning model, and running a Python program to measure the intensity of each cut site autonomously, eliminating the need for manual measurement. Additional investigation of the prevalence of the above mutations in cancer patients will more clearly elucidate their role in tumor formation and progression. Finally, better understanding of the effects of MRE11 mutation on DNA DSB repair provides a new target for more precise treatment of related tumors, and allows for the development of targeted drug therapy for related cancer treatment.

Acknowledgements (Optional): This material was based upon work supported by a gift from Beckman Laser Institute Inc. to LS. The high school students (*) participated in UCSD OPALS internship. Special thanks to Dr. Shu Chien from UCSD Bioengineering and Dr. Lizhu Chen from CorDx Inc. for their donations.

References (Optional): [1] Wang, H., Li, Y., Truong, L.N., Shi,L.Z., Hwang, P.Y.H., He, J., Cho, M.J., Li, H., Negrete, A., Shiloach, J., Berns, M.W., Shen, B., Chen, L. and Wu, X. (2014) CtIP maintains stability at common fragile sites and inverted repeats by end resection-independent endonuclease activity. Mol Cell 54:1012-1021. PMID: 24837675
[2] Duquette ML, Kim J, Shi LZ, Berns MW (2018) LSD1 mediated changes in the local redox environment during the DNA damage response. PLoS ONE 13(8): e0201907. https://doi.org/10.1371/journal.pone.0201907