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

• Project/Area Number: 20K21393
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
• 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
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2021: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000); Fiscal Year 2020: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
• Keywords: DNA一本鎖切断 / トポイソメラーゼI / DNA single-strand breaks / Next-gen sequencing / Genome instability / Cancer / DNA repair

Outline of Research at the Start

1. Development of a methodology to map and quantify in the genome DNA single strand breaks using next-generation sequencing.
I will approach it by two different strategies:
(i) Direct ligation of a labeled sequencing adapter to the end of the SSB.
(ii) SSB labeling by nick translation.
To test the sensitivity and specificity I will utilize CRISPR-Cas9 variants to generate a SSB at defined positions in the genome in vitro and in vivo by inducible expression.

Outline of Final Research Achievements

The main objective of this research is the development of a method to quantify and localize DNA single-strand breaks (SSBs) in the genome. SSBs are discontinuities in one strand of the DNA double helix. It is one of the most frequent damage in the DNA and its mainly produced by endogenous sources, although it can be also the consequence of exposure to UV, irradiation or to genotoxic agents. I have developed a method to quantify and localize DNA single-strand breaks (SSBs) in the genome. After checking the sensitivity detecting induced SSBs in vitro and in vivo I applied it to map the activity of Topoisomerase I in the genome, which creates a SSB as part of its catalytic cycle.

Academic Significance and Societal Importance of the Research Achievements

DNAの一本鎖切断（SSB）は、DNAにおける最も一般的な損傷の一つである。一本鎖切断の蓄積は、癌、神経変性疾患、心不全などの病態に関与している。ゲノム中のSSBsの位置をマッピングする方法を開発することは、この種のDNA損傷の起源と修復、そしてそれらがどのように神経細胞の機能障害や早期老化につながるのか、また癌細胞におけるPARP阻害剤やトポイソメラーゼ阻害剤の作用機序の特徴をよりよく理解するのに役立つであろう。SSBがどのように認識され、修復されるかをよりよく理解することは、将来、がん治療のための改善された治療薬の設計に役立つ可能性がある。

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
  • ISBN: 9781071606438, 9781071606445
  • 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 Issue: 10
  • 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 Issue: 7859 Pages: 440-444
  • DOI: 10.1038/s41586-021-03468-5
  • Peer Reviewed / Int'l Joint Research