Figure 4. ATR regulates the accumulation of RPA at forks via phosphorylation of RPA2 Ser-33.

(A) Western-blot analysis of the expression of RPA2 and RPA2 mutants for RPA2 siRNA and RPA2 siRNA + transfection of myc-tagged wild-type (WT) RPA and S33A, S33D RPA2 mutants.

(B) Overlaid PCNA-RPA-MCM TCF-resolved single-replisome configurations from SMLM images of single cells harboring RPA2 WT and RPA2 phosphor-mutants and under CDC7i treatment conditions. Circle size represents the average density of RPA at each fork within a nucleus.

(C) Quantification of the levels of RPA_fork for the TCF-resolved single replisome configurations shown in (B) reveals that the expression of the phosphor-dead RPA2 S33A results in an accumulation of RPA at forks that is independent of origin firing. Mean values and the 1^st and 3^rd quartile were marked as black and colored bars, respectively, N = 43, 48, 52, 41, 39, and 56 for WT-CDC7i-, S33D-CDC7i-, S33A-CDC7i-, WT-CDC7i+, S33D-CDC7i+, and S33A-CDC7i+, respectively.

(D) Quantification of the abundance ratio between data as indicated. Error bars in log (Abundance Ratio) is the propagated SEM.

(E) Schematic illustration of the regulation of RPA_fork levels by ATR via phosphorylation of RPA2 at Ser33.