CNCC: an analysis tool to determine genome-wide DNA break end structure at single-nucleotide resolution

Abstract

Background: DNA double-stranded breaks (DSBs) are potentially deleterious events in a cell. The end structures (blunt, 3′-and 5′-overhangs) at DSB sites contribute to the fate of their repair and provide critical information concerning the consequences of the damage. Therefore, there has been a recent eruption of DNA break mapping and sequencing methods that aim to map at single-nucleotide resolution where breaks are generated genomewide. These methods provide high resolution data for the location of DSBs, which can encode the type of endstructure present at these breaks. However, genome-wide analysis of the resulting end structures has not been investigated following these sequencing methods. Results: To address this analysis gap, we develop the use of a coverage-normalized cross correlation analysis (CNCC) to process the high-precision genome-wide break mapping data, and determine genome-wide break end structure distributions at single-nucleotide resolution. We take advantage of the single-nucleotide position and the knowledge of strandness from every mapped break to analyze the relative shifts between positive and negative strand encoded break nucleotides. By applying CNCC we can identify the most abundant end structures captured by a break mapping technique, and further can make comparisons between different samples and treatments. We validate our analysis with restriction enzyme digestions of genomic DNA and establish the sensitivity of the analysis using end structures that only exist as a minor fraction of total breaks. Finally, we demonstrate the versatility of our analysis by applying CNCC to the breaks resulting after treatment with etoposide and study the variety of resulting end structures. Conclusion: For the first time, on a genome-wide scale, our analysis revealed the increase in the 5′ to 3′ end resection following etoposide treatment, and the global progression of the resection. Furthermore, our method distinguished the change in the pattern of DSB end structure with increasing doses of the drug. The ability of this method to determine DNA break end structures without a priori knowledge of break sequences or genomic position should have broad applications in understanding genome instability.

DNA fragility at topologically associated domain boundaries is promoted by alternative DNA secondary structure and topoisomerase II activity

“…Detection of DSBs using either purified genomic DNA or fixed nuclei was performed as described ( 9 , 42 , 43 ). Briefly, fixed nuclei were subjected to blunting/A-tailing reactions and Illumina P5 adapter ligation to capture DSB ends.…”

“…This strong correlation confirms that the break mapping procedure does not introduce significant amounts of random DNA breaks that could convert single-stranded nicks into DSBs. The DSB data from GM13069 cells ( 42 ), neural progenitor cells (NPCs) ( 9 ) and HeLa cells ( 44 ) were previously published.…”

Emerging Technologies for Genome-Wide Profiling of DNA Breakage

“…In January of 2020, Szlachta et al presented the first technique for mapping endogenous DSB end structures at single nucleotide resolution (Szlachta et al, 2020 ). This method, known as coverage-normalized cross correlation sequencing (CNCC-seq), is modified from the DSBcapture protocol and creatively employs both the polymerizing and exonuclease activities of a specific DNA Polymerase I (DNApolI).…”