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 through pulse labeling of nascent DNA with distinct thymidine analogs. However, tight sister chromatid cohesion during spreading causes leading and lagging strands to appear as a single fiber, masking strand-specific replication changes that are increasingly recognized as biologically and therapeutically important. Here, we report the unexpected finding that lentiviral infection of human cell lines prior to DNA spreading enables visualization of lagging strand–specific single-stranded DNA gaps arising from defects in Okazaki fragment maturation. Our results suggest that such an effect is transient and independent of viral vectors, encoded sequences, or genome integration. Mechanistically, lentivirus reduces cohesin level on nascent DNA, indicating that transient loosening of sister chromatid cohesion allows daughter strand separation. We propose that pre-exposure to control lentiviruses can be a simple, effective modification to enable the DNA fiber spreading assay to detect strand-specific DNA replication changes and overcome its limitation.