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Significance Repair of chromosome breaks by polymerase theta-mediated end joining (TMEJ) requires short sequence identities in flanking DNA (microhomologies)—a sequence-context constraint that is unique among DNA repair pathways. Though microhomologies have a central role in TMEJ, it has been uncertain whether an organized mechanism to identify them even exists. Using a combination of chromosomal and extrachromosomal substrates, we describe how polymerase theta efficiently locates microhomologies when present, and creates them de novo when absent. We show how this generates a pattern of microhomology-mediated end joining products that is sufficiently distinct from other end joining pathways and that it can be used as a biomarker for TMEJ activity in cancer genomes.

Mechanistic basis for microhomology identification and genome scarring by polymerase theta