qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing.

Area of Science:

• Molecular Biology
• Genetics
• Genomics

Background:

• DNA double-strand breaks (DSBs) are critical DNA lesions associated with genome instability.
• Existing sequencing methods for DSB mapping lack quantitative accuracy and precise genomic localization.
• Understanding physiological DSB relevance requires methods that measure absolute frequencies and locations.

Purpose of the Study:

• To introduce quantitative DSB sequencing (qDSB-Seq), a novel method for precise DSB mapping and quantification.
• To enable measurement of DSB frequencies per cell and their exact genomic coordinates.
• To facilitate accurate comparisons of absolute DSB frequencies across different experimental conditions and samples.

Main Methods:

• Development of qDSB-Seq, integrating site-specific endonuclease-induced spike-in DSBs for internal calibration.
• Utilizing spike-in DSBs to quantify labeled DSBs (e.g., via i-BLESS) with high accuracy.
• Application of qDSB-Seq to measure DSBs induced by radiomimetic drugs, replication stress, and Top1 activity at replication fork barriers.

Main Results:

• qDSB-Seq provides both absolute DSB frequencies per cell and precise genomic coordinates.
• Demonstrated two orders of magnitude differences in DSB frequencies induced by radiomimetic drugs and replication stress.
• Quantified absolute frequencies of Top1-dependent DSBs at natural replication fork barriers.

Conclusions:

• qDSB-Seq overcomes limitations of previous methods, offering quantitative and precise DSB mapping.
• The method allows for accurate comparisons of absolute DSB frequencies across diverse biological contexts and organisms.
• qDSB-Seq significantly advances the study of genome instability and DNA repair mechanisms.