Development of a methodology to map and quantify in the genome DNA single strand breaks.

2020 Fiscal Year Research-status Report

• Project/Area Number: 20K21393
• Research Institution: Kyoto University
• Principal Investigator: Canela Andres 京都大学, 白眉センター, 特定准教授 (90837585)
• Project Period (FY): 2020-07-30 – 2022-03-31
• Keywords: DNA single-strand breaks / Next-gen sequencing / Genome instability / Cancer / DNA repair

Outline of Annual Research Achievements

DNA single-strand breaks (SSBs) are discontinuities in one strand of the DNA double helix that if not repaired can result in genome instability leading to cancer and neurodegenerative diseases. The purpose of the research is the development of a method to quantitatively determine the location of DNA single-strand breaks (SSBs) in the genome. During the fiscal year 2020, I started to set up 2 different approaches to detect SSBs in the genome using next-generation sequencing: direct ligation and nick translation. In the fiscal year 2021, I will evaluate them and apply them to map SSBs in cell lines deficient in SSB repair.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

During the fiscal year 2020:
1. Development of a methodology to map and quantify in the genome DNA single strand breaks using next-generation sequencing. I have made significative advances in this aim setting up the 2 approaches that I will evaluate.

Strategy for Future Research Activity

During the fiscal year 2020:
1. Development of a methodology to map and quantify in the genome DNA single strand breaks using next-generation sequencing.
1.1. I will evaluate which approach is more sensitive and specific using a CRISPR-Cas9 variants to generate a SSB at defined positions in the genome.
1.2. I will quantitatively determine the location of DNA single-strand breaks (SSBs) in the genome in cell lines deficient in SSB repair.

Causes of Carryover

During the fiscal year 2020, although I was able to start setting up the 2 strategies of my research project, I had a delay in the start of the project because of COVID-19.

Research Products

• [Journal Article] END-seq: An Unbiased, High-Resolution, and Genome-Wide Approach to Map DNA Double-Strand Breaks and Resection in Human Cells (2021)
  • Author(s): Wong N, John S, Nussenzweig A, Canela A.
  • Journal Title: Methods in Molecular Biology Volume: 2153 Pages: 9-31
  • DOI: 10.1007/978-1-0716-0644-5_2
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes (2021)
  • Author(s): Huang SN, Michaels SA, Mitchell BB, Majdalani N, Vanden Broeck A, Canela A, Tse-Dinh YC, Lamour V, Pommier Y.
  • Journal Title: Science Advances Volume: 7 Pages: not yet
  • DOI: 10.1126/sciadv.abe0384
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Neuronal enhancers are hotspots for DNA single-strand break repair. (2021)
  • Author(s): Wu W, Hill SE, Nathan WJ, Paiano J, Callen E, Wang D, Shinoda K, van Wietmarschen N, Colon-Mercado JM, Zong D, De Pace R, Shih HY, Coon S, Parsadanian M, Pavani R, Hanzlikova H, Park S, Jung SK, McHugh PJ, Canela A, Chen C, Casellas R, Caldecott KW, Ward ME, Nussenzweig A.
  • Journal Title: Nature Volume: online Pages: not yet
  • DOI: 10.1038/s41586-021-03468-5
  • Peer Reviewed / Int'l Joint Research