Lentivirus enables the detection of strand-specific ssDNA gaps by the DNA fiber spreading assay.

The single-molecule DNA fiber spreading assay is widely used to study DNA replication fork dynamics. However, tight sister chromatid cohesion during DNA spreading causes the leading and lagging strands to collapse into a single fiber, masking strand-specific alterations that are increasingly recognized as biologically and therapeutically important. Here, we report an unexpected finding that lentiviral infection of human cell lines prior to DNA spreading enables visualization of strand-specific single-stranded DNA gaps arising from defects in Okazaki fragment maturation or PrimPol overactivation. Our results suggest that this effect is transient and independent of viral vectors, encoded sequences, or genome integration. Mechanistically, lentivirus transiently reduces cohesin level on nascent DNA, indicating that loosening of sister chromatid cohesion during spreading allows daughter strand separation. We propose that lentiviral pre-exposure can be a simple, effective modification to enable the DNA fiber spreading assay to detect strand-specific DNA replication changes and overcome its limitation.