Single-stranded DNA break detection using an engineered error-prone DNA polymerase

Analysis on whether our DNA is subjected to lesions is important to identify mitogenic substances. Detection of single-stranded DNA breaks (SSBs) has been challenging, due to the lack of appropriate methods. We have engineered a chimeric DNA polymerase, Sloppymerase, that provides the ability to replicate DNA in the absence of one nucleotide. In addition to its polymerase activity, Sloppymerase has a 5'-3' exonuclease activity. We have characterized the activity of Sloppymerase, and utilized it to develop a method for sequence-templated erroneous end-labelling (STEEL-seq) to map SSBs. By omitting one type of nucleotide, e.g., dATP, from the reaction mix, Sloppymerase will introduce mismatches directly downstream the SSBs at positions that should contain deoxyadenosines. The ability to retain sequence information at the end-labelling will ensure that the detected SSBs are bona fide SSBs. We demonstrate that STEEL-seq works with Sanger, Illumina, PacBio and Nanopore sequencing technologies.