FIG 7.

Productive replication is abrogated in HFK-31 cells treated with an inhibitor of Rad51 activity, which reduces HR activity. (A) DNA was harvested from HFK-31 cells at T₀ as well as after differentiation in high-calcium medium for 72 h in the presence of DMSO (DM) or increasing concentrations of the Rad51 inhibitor B02. DMSO was added at a volume equal to that of the 30 μM concentration of B02. DNA was digested with BamHI (noncutter) or HindIII to linearize the viral genome and then analyzed by Southern blotting to examine episome copy number using the HPV31 genome as a probe. Western blot analysis was performed to examine the effect of B02 treatment on Rad51 levels and differentiation (involucrin). (B) Fold change in episome copy number in response to B02 treatment was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Graphed is the average fold change relative to DMSO (DM)–72-h Ca, which is set to 1. Error bars represent means ± standard errors. Statistics were assayed using a two-tailed t test. *, P < 0.01. (C) Schematic of the DR-GFP reporter used to monitor HR activity. SceGFP is a modified GFP gene containing an I-SceI restriction site and in-frame termination codons. The iGFP fragment can be used to repair the I-SceI-induced DSB through HR, resulting in GFP expression. (D) HFK-31 cells containing a chromosomally integrated homologous recombination reporter cassette (DR-GFP) were transduced with I-SceI-containing lentivirus particles to induce a DSB within the DR-GFP reporter cassette. DMSO (DM) or increasing concentrations of B02 were added 24 h after transduction with I-SceI. Forty-eight hours after addition of B02, cells were fixed and analyzed for GFP expression by flow cytometry as a measurement of HR repair. Shown is the fold change in percent GFP relative to the untreated (UT) sample. Graphed values are averages of values from five independent experiments, with error bars representing means ± standard errors. Statistics were assayed using a two-tailed t test. *, P < 0.05. Ca, calcium; WB, Western blot.