Fig. 1. RPA interacts with RNaseH1 and colocalizes with RNaseH1 and R loops.

(A) RNaseH1-GFP (the mitochondrial localization signal of RNaseH1 was removed) or SFB-GFP was expressed in HEK293T cells and immunoprecipitated using anti-GFP antibody. Endogenous RPA32 associated with RNaseH1-GFP was detected by Western blot. Cell lysates (1%) were loaded as input. (B) RNaseH1-GFP was expressed in HEK293T cells either alone or in combination with SFB-RPA70 or SFB-RPA32 and immunoprecipitated using anti-Flag antibody conjugated to agarose beads. Indicated tagged proteins were detected by Western blot. (C) HeLa-derived RNaseH1 inducible cells were transfected with control or AQR siRNA and cultured for 60 h. RNaseH1 expression was induced by doxycycline for 48 h. R loop levels in individual cells were analyzed using the S9.6 antibody. Representative images for each condition are shown. S9.6 intensity quantification and expression of RNaseH1-GFP are shown in Fig. S1E–F. (D-G) Pair-wise colocalization of RNaseH1^D210N-GFP, endogenous RPA32 (or RPA32 p-S33), and R loops (S9.6 foci) were analyzed in AQR knockdown cells as indicated in D, E, and F. RNaseH1^D210N-GFP was induced in D and E similar to C. A summary of the three-way colocalization is shown in G. (H-I) HeLa-derived RNaseH1^D210N-GFP cells were induced with doxycycline for 48 h prior to fixation and fragmented. Chromatin was immunoprecipitated using anti-GFP (in H) and anti-RPA32 (in I). The association of RNaseH1^D210N and RPA with the indicated loci was analyzed by ChIP-qPCR (n=3). Asterisks indicate p<0.05. (J) HeLa cells were transfected with control or AQR siRNA and cultured for 48 h. S-phase and non-S-phase cells were distinguished by 30-min EdU labeling. R loop levels in individual cells were analyzed with the S9.6 antibody (n=50). Red bars represent the mean S9.6 intensities of the indicated cell populations. (K) HeLa-derived cells were transfected with control or AQR siRNA and induced to express RNaseH1-GFP as indicated. S-phase and non-S-phase cells were distinguished by EdU labeling as in J. Levels of RPA32 p-S33 in individual cells were analyzed with the RPA p-S33 antibody (n>100). Red bars represent the mean RPA32 p-S33 intensities of the indicated cell populations. See also Fig. S1.