Figure 4.

Gapped Forks and DNA-RNA Hybrid Accumulation at the PDC1-TRX1 Locus in Sen1-Depleted Cells

(A) Scheme, scaled on a representative 2D gel sen1 cells profile (Figure 3E), showing replication fork progression across the SphI restriction fragment containing the PDC1-TRX1 genes. The putative intermediates are shown. RNA (dashed blue line) in DNA-RNA hybrids was removed by RNase treatment prior the 2D gel analysis. Yellow lines mark the gapped regions on Ys intermediates predicted on the base of the sen1 2D gel profiles (see text for details).

(B) RNases and Mung Bean nuclease sensitivity analysis on sen1 aberrant replication intermediates at 150 min in HU.

(C and D) (C) DNA-RNA hybrids and RFA1-PK (Rfa1) (D) accumulation at PDC1 locus by ChIP followed by qPCR analysis in WT and GAL::URL-HA-SEN1 cells treated as in Figure 3E. Data represent the mean ±SD of three independent experiments. See also Figure S4.