Figure 5
From: RNA-cDNA hybrids mediate transposition via different mechanisms
Increased Ty1 retromobility in rnh1∆ rad27∆ but not rnh1∆ rnh201∆ mutants is dependent on Rad52. (a) A schematic of canonical (integrase) and non-canonical (HR) mechanisms of Ty1 mobility. Ty1 can mobilize by HR through: (i) recombination anywhere along a Ty1 sequence, or (ii) recombination with a solo LTR. (b) Effect of Rad52 loss on Ty1his3AI retromobility (one-way ANOVA followed by Sidak’s post-hoc test). Mean ± SD; n = 5. (c) Effect of Rad52 loss on Ty1 mRNA levels (one-way ANOVA followed by Sidak’s post-hoc test). TYA (left) and Tyhis3 (right) were amplified and ACT1 was used as an internal control. Mean ± SD; n = 3. (d) Effect of Rad52 loss on Ty1 Gag levels determined by western blot. Act1 is presented as a loading control. (e) Effect of Rad52 loss on Ty1 cDNA levels as detected by Southern blot. M = marker. (f) Effect of Rad52 loss on levels of RNA-cDNA hybrids at TYA (top) and TYB (bottom; one-way ANOVA followed by Sidak’s post-hoc test). Mean ± SD; n = 3. (g) Sequential ChIP assessing the co-enrichment of RNA-cDNA hybrids and ssDNA at Ty1 followed by qPCR directed at TYA (top) and TYB (bottom). Results are calculated as % S9.6 input normalized to CUP1 and presented relative to wild-type. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc test. Mean ± SD; n = 3. (a–g) *p < 0.05; **p < 0.01; n.s. = not statistically significant.
