Precise mapping of single-stranded DNA breaks by sequence-templated erroneous DNA polymerase end-labelling

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Accurate detection of DNA lesions is critical for identifying mutagenic agents.
• Current methods for detecting single-stranded DNA breaks (SSBs) lack precision.

Purpose of the Study:

• To develop a precise method for mapping single-stranded DNA breaks (SSBs).
• To engineer a DNA polymerase capable of facilitating SSB detection.

Main Methods:

• Developed sequence-templated erroneous end-labelling sequencing (STEEL-seq) for SSB mapping.
• Engineered a chimeric DNA polymerase, Sloppymerase, for error-prone replication.
• Utilized nucleotide omission to induce specific mismatches downstream of SSBs.

Main Results:

• STEEL-seq accurately maps SSBs by introducing a detectable mismatch pattern.
• Demonstrated STEEL-seq compatibility with Sanger, Illumina, PacBio, and Nanopore sequencing.
• Quantified human genome SSB/base pair frequency between 0.7-3.8 × 10^-6.
• Observed enrichment of SSBs in active human promoter regions.

Conclusions:

• STEEL-seq offers a precise and versatile tool for mapping SSBs across various sequencing platforms.
• The engineered Sloppymerase is key to the high fidelity of the STEEL-seq method.
• The findings provide insights into the genomic distribution of SSBs, particularly in regulatory regions.