PRIMPOL competes with RAD51 to resolve G-quadruplex-induced replication stress via its interaction with RPA

Abstract

PRIMPOL (primase-polymerase) is a recently discovered DNA primase-polymerase involved in DNA damage tolerance and replication stress response in eukaryotic cells. However, the detailed mechanism of the PRIMPOL response to replication stress remains elusive. Here, we demonstrate that replication-related factors, including replication protein A (RPA), regulate the accumulation of PRIMPOL in subnuclear foci in response to replication stress induced by replication inhibitors. Moreover, PRIMPOL works at G-quadruplexes (G4s) in human cells to resolve the replication stress induced by G4s. The formation of PRIMPOL foci persists throughout the cell cycle. We further demonstrate that PRIMPOL competes with RAD51 to resolve G4-induced replication stress. In conclusion, our results provide novel insight into the mechanism of PRIMPOL in G4s to resolve replication stress and competition between PRIMPOL (repriming)- and RAD51 (fork reversal)-mediated pathways, which indicates a new strategy to improve the tumor response to DNA-damaging chemotherapy by targeting the PRIMPOL pathway.

Keywords: G-quadruplex; PRIMPOL; RAD51; RPA; replication stress.

Figure 1

PRIMPOL interacts with replication-related factors in response to replication stress (A) Silver staining of HEK 293T cells transiently transfected with FLAG-vector or FLAG-PRIMPOL. (B) The proteins associated with PRIMPOL were identified by mass spectrometry, as indicated. (C) Co-IP assay indicated the interaction between PRIMPOL and RPA2. (D) Immunofluorescence (IF) assay was carried out using anti-RPA2 (green) and anti-PRIMPOL (red) antibodies in HeLa and U2OS cells. HeLa cells were treated with HU (2 mM) for 24 h. DAPI (blue). The colocalized signals are shown in enlarged images. HU, hydroxyurea. (E) Quantification of the number of colocalizations in PRIMPOL foci. **** P<0.0001.

Figure 2

PRIMPOL is required to resolve G4 structure-induced replication stress (A) Quantification of the number of G4 foci in HeLa, HEK 293T, and U2OS cells in Supplementary Figure S2A. The cells were treated with TMPyP4 (50 μM) or PDS (10 μM) for 24 h. (B) Representative images of PRIMPOL (red) and G4 (green) colocalization in HeLa and U2OS cells. White arrows indicate colocalizations. The cells were treated with PDS (10 μM) for 24 h. (C) Quantification of the colocalization of PRIMPOL and G4 in control and PDS-treated cells. (D) Representative IF showing the nuclear foci formation of PRIMPOL in HeLa, HEK 293T, and U2OS cells. The cells were treated with TMPyP4 (50 μM) or PDS (10 μM) for 24 h. DAPI (blue); PRIMPOL (green). (E,F) Quantification of the percentage of nuclei with PRIMPOL foci and the number of PRIMPOL foci per cell. Approximately 200 cells were analysed. (G) The expression of PRIMPOL is shown in the PRIMPOL-knockdown clones of HeLa and U2OS cells. (H) Representative images of G4 foci in control (scramble) and PRIMPOL-knockdown HeLa and U2OS cells. DAPI (blue); G4 (red). (I) Quantification of the number of G4 foci per cell. (J,K) Approximately 1000 HeLa cells and 500 U2OS cells were seeded. Cell viability was assessed for 5 days by CCK8 assays. * P<0.05, ** P<0.001, *** P<0.001, **** P<0.0001.

Figure 3

The formation of PRIMPOL foci persists throughout the cell cycle (A) U2OS cells were synchronized to G1, S and G2 phases respectively. Representative FACS profile by PI staining. (B) PRIMPOL and EdU were detected by IF in synchronized cells. White arrows indicate colocalizations. (C) Quantification of the number of PRIMPOL foci in the synchronized cells containing PRIMPOL foci. (D) Quantification of the colocalization of PRIMPOL and EdU per cell. (E) Quantification of the percentage of nuclei with ≥5 PRIMPOL-EdU colocalized foci.** P<0.001, **** P<0.0001.

Figure 4

PRIMPOL competes with RAD51 to resolve G4-induced replication stress (A) Cells were transiently transfected with control, RPA siRNA, or RAD51 siRNA. Western blot analysis showing the knockdown of RPA and RAD51 in HeLa, HEK 293T, and U2OS cells. (B) Representative images showing the formation of PRIMPOL foci in RPA-knockdown, RAD51-knockdown, and control cells. DAPI (blue); PRIMPOL (red). (C) Quantification of the number of PRIMPOL foci per cell. More than 100 cells were analysed. (D) Representative images of G4 foci in the control (NC) and RPA- or RAD51-knockdown HeLa, HEK 293T, and U2OS cells. DAPI (blue); G4 (red). (E) Quantification of the number of G4 foci per cell. (F) Representative images of RAD51 (green) and G4 (red) colocalizations in control (scramble) and PRIMPOL-knockdown HeLa and U2OS cells. White arrows indicate colocalizations. (G) Quantification of the colocalization of PRIMPOL and G4 per cell. PRIMPOL and RAD51 were detected by IF in U2OS cells. DAPI (blue); PRIMPOL (red); RAD51 (green). * P<0.05, ** P<0.001, *** P<0.001, **** P<0.0001.