I-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks

Anna Biernacka,Yingjie Zhu,Philippe Pasero,Benjamin Pardo,Magdalena Skrzypczak,Krzysztof Ginalski,Romain Forey,Nicola Crosetto,Maga Rowicka,Marta Grzelak,Jules Nde,Abhishek Mitra,Andrzej Kudlicki

doi:10.1038/s42003-018-0165-9

Abstract

Maintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

Highlights

Maintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs)
We computationally analyzed patterns of DSBs detected by i-BLESS to find signatures distinguishing genuine breaks from artifacts and observed a high periodicity of the background signal, with a period of 162 bp, which corresponds to the typical distance between nucleosomes in S. cerevisiae[17] (Supplementary Fig. 1a)
After comprehensive studies, we identified a range of parameters optimal for highly specific DSB detection, among which intensive proteinase K treatment (50 μg mL−1 overnight at 50 °C) turned out to be crucial (Fig. 1b)

Summary

Introduction

Maintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Several next-generation sequencing methods have been recently developed to label DSBs directly and genome-wide in mammalian cells[9,10,11], starting with our BLESS (Breaks Labeling, Enrichment on Streptavidin and next-generation Sequencing) method[12] These techniques cannot be applied to detect DSBs in yeast. One commonly used strategy to overcome this issue is encapsulation of cells in agarose, which protects DNA from mechanical damage This approach was used by Mimitou et al in S1-Seq[13] to label DSBs resulting from end resection in yeast. We present i-BLESS (immobilized-BLESS), a new method for direct in situ genome-wide DSB labeling in agarose beads, optimized for yeast, but in principle applicable to all ( small) cells.

Methods

Results

Conclusion