Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells

Abstract

PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.

Keywords: BRCA1; BRCA2; DNA damage tolerance; DNA replication; PRIMPOL; genome stability; post-replicative repair; replication stress; ssDNA gaps; translesion synthesis polymerases.

Figure 1.. Suppression of fork reversal and overexpression PRIMPOL lead to ssDNA gaps.

(A) Top, schematic of the DNA fiber assay with the S1 nuclease. Bottom, dot plot and median of CldU tract lengths in U2OS cells overexpressing WT- or CH-PRIMPOL + 150 μM cisplatin ± S1 nuclease (n=3). ns, non-significant, ****p < 0.0001. (B) Dot plot and median of CldU tract lengths in wild-type or SMARCAL1KO U2OS cells ± 150 μM cisplatin ± siCTRL (control siRNA) or siPRIMPOL ± S1 nuclease (n=3). ns, non-significant, ****p < 0.0001. (C) Dot plot and median of CldU tract lengths in SIOD cells or SIOD cells complemented with wild-type SMARCAL1 ± 150 μM cisplatin ± S1 nuclease (n=3). ns, non-significant, ****p < 0.0001. (D) Dot plot and median of CldU tract lengths in U2OS cells ± PARPi ± 150 μM cisplatin ± siCTRL or siPRIMPOL ± S1 nuclease (n=3). ns, non-significant, ****p < 0.0001. (E) Left, representative electron micrograph of a reversed replication fork. Right, percentage of reversed replication forks in wild-type, SMARCAL1KO, and PARPi-treated U2OS cells ± 150 μM cisplatin (n=3). Statistics: unpaired t test; **p < 0.01, ***p < 0.001. (F) Left, representative electron micrograph of a replication fork with an internal ssDNA gap behind the fork (daughter strand gap), indicated by the red arrow. Right, percentage of replication forks with daughter strand gaps in wild-type, SMARCAL1KO, and PARPi-treated U2OS cells ± 150 μM cisplatin (n=3). P: parental strand, D: daughter strand, R: reversed arm. Statistics: unpaired t test; *p < 0.05, **p < 0.01. See also Figure S1.

Figure 2.. PRIMPOL-dependent gaps are repaired in S and G2 phase.

(A) Top, schematic of the DNA fiber assay with the S1 nuclease to determine the kinetics of ssDNA gap repair. Bottom, dot plot and median of CldU tract lengths in PRIMPOL-OE (overexpressing) U2OS cells ± 150 μM cisplatin treated with the S1 nuclease at the indicated times (n=3). ns, non-significant, *p < 0.05, ****p < 0.0001. (B) Top, schematic for kinetic flow cytometry analysis of replicating cells ± 150 μM cisplatin. Bottom, cell cycle histograms (stained with DAPI to assess DNA content) of EdU-positive PRIMPOL-OE U2OS cells ± 150 μM cisplatin, collected at the indicated times. (C) Top, schematic for post-replication repair (PRR) assay. Bottom, representative images of high-density and low-density PRR tracts. (D) Dot plot and median of PRR densities in mock-transfected or PRIMPOL-OE U2OS cells ± 150 μM cisplatin (n=3). ns, non-significant, ****p < 0.0001. (E) Dot plot and median of PRR densities in wild-type or SMARCAL1KO U2OS cells ± 150 μM cisplatin ± siCTRL or siPRIMPOL (n=3). ns, non-significant, ****p < 0.0001. (F) Dot plot and median of PRR densities in SIOD cells or SIOD cells complemented with SMARCAL1 ± 150 μM cisplatin (n=3). ns, non-significant, ****p < 0.0001. (G) Dot plot and median of PRR densities in U2OS cells ± PARPi ± 150 μM cisplatin (n=3). ns, non-significant, ****p < 0.0001. See also Figure S2.

Figure 3.. Gap filling in G2 is dependent on RAD18, PCNA monoubiquitination and REV1-POLζ.

(A) Dot plot and median of PRR densities in wild-type or PRIMPOL-OE U2OS cells + 150 μM cisplatin ± siCTRL or siRAD18 or siUBC13 (n=3). ns, non-significant, ****p < 0.0001. (B) Dot plot and median of PRR densities in wild-type or SMARCAL1KO U2OS cells + 150 μM cisplatin ± siCTRL or siRAD18 (n=3). ns, non-significant, ****p < 0.0001. (C) Dot plot and median of PRR densities in wild-type HEK293T cells or HEK293T cells expressing the PCNA K164R mutant ± PRIMPOL-OE + 150 μM cisplatin (n=3). ns, non-significant, ****p < 0.0001. (D) Dot plot and median of PRR densities in wild-type or PRIMPOL-OE U2OS cells + 150 μM cisplatin ± siCTRL or siPOLηi or siREV3L (n=3). ns, non-significant, ****p < 0.0001. (E) Top, schematic of PRR assay treated with REV1 inhibitor (REVi, JH-RE-06). Bottom, dot plot and median of PRR densities in wild-type or PRIMPOL-OE U2OS cells + 150 μM cisplatin ± 2 μM REV1i (n=3). ns, non-significant, ****p < 0.0001. (F) Dot plot and median of PRR densities in wild-type or SMARCAL1KO U2OS + 150 μM cisplatin ± 2 μM REV1i (n=3). ns, non-significant, ****p < 0.0001. See also Figure S3.

Figure 4.. REV1-POLζ, UBC13, and RAD51 mediate gap filling in S phase.

(A) Top, schematic of PLA assay. Bottom, representative images of RAD18-EdU PLA foci (red) wild-type and SMARCAL1KO U2OS cells ± 150 μM cisplatin. Cyclin B1 is used as a marker for G2. White dashes outline nuclear contours as detected by DAPI staining. (B) Percentage of cells in G2 (cyclin B1+) with >3 RAD18-EdU PLA foci (mean ± SEM) in wild-type and SMARCAL1KO U2OS cells (n=5). *p < 0.05. (C) Percentage of cells in G2 with >3 REV1-EdU PLA foci (mean ± SEM) in wild-type and SMARCAL1KO U2OS cells (n=3). ***p < 0.001. (D) Percentage of cells in G2 with >3 UBC13-EdU PLA foci (mean ± SEM) in wild-type and SMARCAL1KO U2OS cells (n=3). ns, non-significant. (E) Top, schematic of DNA fiber with S1 nuclease assay at the indicated times after cisplatin treatment. Bottom, dot plot and median of CldU tract lengths in SMARCAL1KO U2PS cells ± siCTRL or siREV3L, ± S1 nuclease at 0 hours or + S1 at 8 and 16 hours post a 1 hour treatment with 150 μM cisplatin (n=2). ***p < 0.001, ****p < 0.0001. (F) Top, schematic of DNA fiber with S1 nuclease assay with REV1 inhibitor (REV1i, JH-RE-06). Bottom, dot plot and median of CldU tract lengths in SMARCAL1KO U2OS cells ± 2 μM REV1i, ± S1 nuclease at 0 hours or + S1 at 8 and 16 hours post a 1 hour treatment with 150 μM cisplatin (n=2). *p < 0.05, ****p < 0.0001. (G) Top, schematic of DNA fiber with S1 nuclease assay at the indicated times after cisplatin treatment. Bottom, dot plot and median of CldU tract lengths in SMARCAL1 KO U2OS cells ± siCTRL or siUBC13, ± S1 nuclease at 0 hours or + S1 at 8 and 16 hours post a 1 hour treatment with 150 μM cisplatin (n=3). ns, non-significant, **p < 0.01, ****p < 0.0001. (H) Top, schematic of DNA fiber with S1 nuclease assay with RAD51 inhibitor (RAD51i, B02). Bottom, dot plot and median of CldU tract lengths in SMARCAL1KO U2OS cells ± 27 μM RAD51i, ± S1 nuclease at 0 hours or + S1 at 8 and 16 hours post a 1 hour treatment with 150 μM cisplatin (n=2). ns, non-significant, ***p < 0.001, ****p < 0.0001. (I) Top, schematic of DNA fiber with S1 nuclease assay at the indicated times after cisplatin treatment. Bottom, dot plot and median of CldU tract lengths in PRIMPOL-OE RAD51^+/+ and RAD51 T131P cells ± S1 nuclease at 0, 8 and 16 hours post a 1 hour treatment with 150 μM cisplatin (n=3). ns, non-significant, ****p < 0.0001. See also Figure S4.

Figure 5.. BRCA1 promotes gap filling by limiting MRE11 activity.

(A) Dot plot and median of PRR densities in PRIMPOL-OE U2OS cells treated with increasing concentrations of cisplatin (150, 300, and 600 μM) ± siCTRL or siBRCA1 (n=3). ns, non-significant, *p < 0.05, ****p < 0.0001. (B) Top, expression of PRIMPOL after transfection of V5-tagged PRIMPOL in UW and UW+BRCA1 cells. Bottom, dot plot and median of PRR densities in UW and UW+BRCA1 cells ± PRIMPOL-OE + 150 μM cisplatin (n=3). ns, non-significant, ****p < 0.0001. (C) Top, expression of SMARCAL1 after siRNA (siCTRL or siSMARCAL1) knockdown in UW and UW+BRCA1 cells. Bottom, dot plot and median of PRR densities in UW and UW+BRCA1 cells ± siCTRL or siSMARCAL1 + 150 μM cisplatin (n=3). ns, non-significant, ****p < 0.0001. (D) Top, schematic of PRR assay with MRE11 inhibitor (mirin). Bottom, dot plot and median of PRR densities in SMARCAL1KO U2OS cells ± 150 μM cisplatin ± siCTRL or siBRCA1 ± 50 μM mirin (n=3). ns, non-significant, ***p < 0.001, ****p < 0.0001. (E) Top, schematic of kinetic fiber with S1 nuclease and MRE11 inhibitor (mirin). Bottom, dot plot and median of CldU tract lengths in SMARCAL1KO cells ± siCTRL or siBRCA1 ± 50 μM mirin ± S1 nuclease at 0 or + S1 at 8 and 16 hours post a 1 hour treatment with 150 μM cisplatin. ns, non-significant, *p < 0.05, ****p < 0.0001. See also Figure S5.

Figure 6.. Inhibition of gap filling leads to DNA double-stranded breaks and G1-specifc 53BP1 nuclear bodies.

(A) Representative images of comets in wild-type and SMARCAL1KO U2OS cells ± siCTRL or siREV3L ± 5 μM cisplatin. (B) Top, schematic of comet assay. Bottom, dot plot and median of tail moments in wild-type and SMARCAL1KO U2OS cells ± siCTRL or siREV3L ± 5 μM cisplatin (n=3). ns, non-significant, ****p < 0.0001. (C) Representative images of 53BP1 nuclear bodies in G1 in wild-type and SMARCAL1KO U2OS cells ± siCTRL or siREV3L ± 5 μM cisplatin. (D) Top, schematic of experiment. Bottom, dot plot and median of G1-specific 53BP1 bodies in wild-type and SMARCAL1KO U2OS cells ± siCTRL or siREV3L ± 5 μM cisplatin (n=3). ****p < 0.0001. (E) Dot plot and median of tail moments in wild-type and SMARCAL1KO U2OS cells ± siCTRL or siBRCA1 ± 5 μM cisplatin (n=2). ns, non-significant, ****p<0.0001. (F) Dot plot and median of G1-specific 53BP1 bodies in wild-type and SMARCAL1 KO U2OS cells ± siCTRL or siBRCA1 ± 5 μM cisplatin (n=3). ****p < 0.0001. (G) Dot plot and median of G1-specific 53BP1 bodies in wild-type and SMARCAL1KO U2OS cells ± siCTRL or siBRCA1 ± 2 μM REV1i + 5 μM cisplatin (n=2). ns, non-significant, ****p < 0.0001. See also Figure S6.

Figure 7.. Inhibition of gap filling negatively affects cell survival.

(A) Cell survival of wild-type and SMARCAL1KO U2OS cells ± siCTRL or siBRCA1 6 days after acute (1 hour) treatment with the indicated doses of cisplatin (means ± SEM) (n=3). **p < 0.01, ***p < 0.001. (B) Cell survival of wild-type and SMARCAL1KO U2OS cells ± siCTRL or siREV3L 6 days after acute (1 hour) treatment with the indicated doses of cisplatin (means ± SEM) (n=5). *p < 0.05, **p < 0.01. (C) Cell survival of UW and UW+BRCA1 cells ± indicated chronic doses (6 days) of PARPi (Olaparib), cisplatin, and REV1i (JH-RE-06) (n=3). ns, non-significant, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Working model for the mechanisms of gap filling following PRIMPOL repriming. In G2, RAD18 promotes PCNA monoubiquitination at K164, which, in turn, recruits the REV1-POLζ complex to fill gaps by TLS. UBC13 and RAD51 are instead required for gap filling in S suggesting that these factors mediate a TS mechanism to fill gaps in S. REV1 and POLζ are also required for gap filling in S phase, suggesting that they either participate in the same pathway or that TLS is active both in S and G2 phase. BRCA1 and BRCA2 protect ssDNA gaps from aberrant MRE11 activity in both S and G2 phase. See also Figure S6.