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

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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.