Detection of Single-Stranded DNA Gaps Reveals a Functional Biomarker of Therapeutic Response.

Abstract

Single-stranded DNA (ssDNA) gaps represent an underappreciated vulnerability that directly shapes therapeutic response in BRCA-deficient models. Current clinical biomarkers remain anchored to the double-strand break (DSB) centric model of homologous recombination deficiency (HRD), and there are currently no tools available to detect replication-associated gaps in the clinical setting. Here, we developed a scalable, clinically adaptable assay that enables real-time, drug-free detection of gaps. This assay demonstrated that gaps are intrinsically elevated in BRCA-deficient cells independent of exogenous DNA damage. These gaps arose during DNA replication and were generated through PRIMPOL-dependent repriming at abasic sites and nuclease-mediated processing. Across diverse cell line models, PARP inhibitor (PARPi) sensitivity was associated with gap burden irrespective of BRCA status, extending the relevance of this vulnerability beyond canonical BRCA-mutant contexts. Integration with AI-driven image analysis enabled automated, unbiased quantification of gap levels in xenograft tumor specimens. In BRCA-deficient xenografts, elevated gap levels were associated with PARPi sensitivity, supporting their translational relevance in relation to therapeutic response. Together, these findings establish ssDNA gaps as a real-time functional readout associated with chemotherapy response and introduce an assay with potential for future clinical translation.