Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery - PubMed (original) (raw)

Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery

Anar K Murphy et al. J Cell Biol. 2014.

Abstract

Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress.

© 2014 Murphy et al.

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Figures

Figure 1.

Figure 1.

RPA phosphorylation stimulates DNA synthesis during stress. (A) RPA2 phosphorylation mutants. The seven known human RPA2 phosphorylation sites are indicated by underlining, with the known or putative kinases shown. WT-RPA2, PIKK_A-RPA2 (double T21A/S33A mutation in the two PIKK sites), and Cdk_A-RPA2 (double S23A/S29A mutation in the two Cdk sites) variants were inducibly expressed from U2-OS stable clones. These three RPA2 variants and the PIKK_D (T21D/S33D mutation) and S4A/S8A mutants were transiently expressed in RPE cells. (B) Schematic of IdU/CldU labeling. (C) Representative fibers labeled with IdU (red) and CldU (green) from cells in which the endogenous RPA2 subunit was replaced with ectopic WT-RPA2, PIKK_A-RPA2, or Cdk_A-RPA2, as indicated. Images were resized to normalize IdU lengths, and the images were sorted so that molecules with the shortest CldU tracts were at the top and longest were at the bottom. For each set of tracts, the white line indicates the position of the IdU–CldU transition. These data indicate that cells replaced with WT-RPA2 have longer CldU tracts compared with cells replaced with PIKK_A- or Cdk_A-RPA2. Bar, 20 µm. (D) RPA2 phosphorylation significantly stimulates fork movement under replication stress conditions but not under unperturbed conditions. Quantitation of fork movement, expressed as the replication fork rate. The fork rate for cells replaced with WT-RPA2 was set at 1.5 kb/min. **, P < 0.01, relative to the fork rate of HU-treated cells replaced with WT RPA. Error bars indicate SEMs. (E) Schematic of [3H]TTP labeling involving either an early or late 90-min labeling period in the presence of 1 mM HU. (F) Mutation of RPA2 PIKK or Cdk phosphorylation sites primarily causes a general slowdown in replication fork movement. Endogenous RPA2 was replaced with ectopic WT-, PIKK_A-, or Cdk_A-RPA2. Two parallel batches of the appropriate replaced cell line were either (early) incubated in HU (15 min) and then HU and [3H]TTP for 90 min or (late) incubated in HU (105 min) and then HU and [3H]TTP for 90 min. The experiment was performed in triplicate. The amount of [3H]TTP incorporated into DNA in the early and late labeling periods was quantitated (see Materials and methods), and the value of [3H]TTP incorporated early/[3H]TTP incorporated late was plotted. The data are expressed as means ± SD.

Figure 2.

Figure 2.

Loss of RPA phosphorylation causes defective recovery of DNA replication forks after replication stress. (A) Schematic of fork labeling procedure. Replicating DNA molecules were first labeled with IdU during 3-h incubation under replication stress conditions followed by a 50-min recovery period in which DNA was labeled with CldU. (B) Cells expressing RPA2 mutated at the PIKK or Cdk sites show defective recovery of fork rate after 3-h treatment with 1 mM HU. (C) Defective RPA phosphorylation causes slower fork rate recovery after 3-h treatment with 30 µM aphidicolin (APH). *, P < 0.05; **, P < 0.01, relative to WT-RPA2 values. Error bars indicate SEMs.

Figure 3.

Figure 3.

Specific RPA phosphorylation sites are important for replication fork movement during replication stress. (A) Western blot showing efficient replacement of RPA2 by transient transfection. Lysates, prepared 72 h after siRNA transfection, were analyzed by Western blotting for RPA2 and β-actin. For the RPA2 blot, note that both the endogenous (Endo) and Myc-tagged species are seen. Black lines indicate that intervening lanes have been spliced out. (B) An S4A/S8A-RPA2 mutant does not have significant effects on fork movement during replication stress conditions. RPE cells were transiently transfected with the various RPA2 expression constructs and, on the next day, transfected with an siRNA selective for the endogenous RPA2 mRNA. Cells were labeled as shown in the schematic, and fibers were then prepared and imaged. Fork rates are shown in comparison to the rates determined in unperturbed cells replaced with WT-RPA2 (set at 1.5 kb/min). **, P < 0.01, relative to WT-RPA2 value for the same condition. The data are expressed as means ± SEM. (C) The PIKK and Cdk mutants, in contrast to the S4A/S8A mutant, causes defects in fork recovery. RPE cells were replaced with the RPA2 variants and then labeled with CldU and IdU as shown in the schematic. Fork rates were calculated and presented as described for B.

Figure 4.

Figure 4.

Hyperphosphorylation of RPA causes defects in fork movement both during replication stress and the recovery from stress. (A) Western blot analysis showing efficient knockdown of the PP4R2 subunit in U2-OS cells. Cells were transfected with a specific siRNA against PP4R2 or negative (Neg) control siRNA. After transfection (72 h), lysates were prepared and analyzed by Western blotting for PP4R2 or β-actin (loading control). (B) PP4R2 knockdown stimulates RPA phosphorylation. After control or PP4R2 knockdown as in A, cells were treated with 1 µM CPT for various times (as indicated). Cell lysates were then prepared and subjected to Western blot analysis for RPA2. The basal (B; nonphosphorylated) and hyperphosphorylated (H) RPA2 species are indicated. (C) Diagram of the fork labeling procedure used to examine the effect of knockdown of the PP4R2 phosphatase subunit. Two distinct fiber-labeling experiments were performed to examine fork movement during unperturbed conditions and replication stress induced by 1 mM HU treatment (experiment [exp.] 1) and during replication stress and the recovery from stress (experiment 2). (D) Deregulation of RPA phosphorylation causes defects in replisome progression during replication stress and recovery from stress. Fork rates during replication stress (sepia) were the mean rates determined in the first and second experiments (outlined in C). **, P < 0.01, relative to fork rates determined in cells treated with the control siRNA. Error bars indicate SEMs.

Figure 5.

Figure 5.

RPA phosphorylation facilitates proper nuclear localization of both PALB2 and BRCA2. (A) RPA and PALB2 show significant colocalization during replication stress. U2-OS cells, transiently transfected with a GFP-PALB2 expression vector, were mock treated or treated with 1 mM HU for 3 h. Cells were then detergent extracted to remove the soluble fraction of the GFP-PALB2 and RPA pools and fixed. After staining for RPA2, cells were imaged for GFP-PALB2, RPA2, and DAPI. (B and C) Mutation of RPA2 phosphorylation sites reduces GFP-PALB2 nuclear retention during replication stress. Cells in which endogenous RPA2 was replaced with ectopic WT-, PIKK_A, or Cdk_A-RPA2 were transfected with a GFP-PALB2 expression vector. Cells were treated with 1 mM HU for 3 h and then imaged as described in A. Images were quantitated using ImageJ (National Institutes of Health), and the relative amount (rel. amt) of PALB2 nuclear staining is shown. Error bars indicate SDs. (D) Mobility of PALB2 under replication stress conditions is significantly higher in cells replaced with either the Cdk_A- or PIKK_A-RPA2 mutant. FRAP analysis of PALB2 mobility is shown, with the data corrected for acquisition bleaching. Prebleach data were used to determine SDs. The calculated _t_1/2 of recovery is shown below the plot. Each combination of RPA2 variant and condition tested was repeated five to seven times. (E) Mutation of RPA phosphorylation sites causes loss of BRCA2–RPA colocalization during replication stress. U2-OS cells were treated with 1 mM HU for 3 h and then extracted and fixed. Cells were stained for BRCA2, RPA2, and DAPI and imaged. (F) PALB2 and BRCA2 are each in close proximity to RPA in cells, particularly after replication stress. U2-OS cells, either transfected with GFP-PALB2 (two left images) or not transfected (two right images), were either mock treated or incubated with 1 mM HU for 3 h. Cells were analyzed for proximal association of GFP-PALB2 and RPA, or BRCA2 and RPA, using the Duolink immunoassay. In brief, the Duolink assay involves two different DNA-conjugated secondary antibodies that, when adjacent to each other, support formation of a circular DNA molecule that can be amplified by a rolling circle DNA replication. The DNA product is then subsequently detected with a complementary and fluorescently labeled oligonucleotide (Söderberg et al., 2006), giving the observed red dots. Bars: (A) 10 µm; (B, E, and F) 15 µm.

Figure 6.

Figure 6.

RPA phosphorylation stimulates recruitment of PALB2 to ssDNA. (A) Phosphorylation of RPA by Cdk. RPA bound to a dT90 substrate was either mock treated (lane 1) or incubated (lane 2) with cyclin B–Cdk1. (B) PALB2 and RPA interact in vitro. (left) The relative amounts of PALB2 recovered, corrected for RPA2 levels, are shown under the PALB2 blot. (right) The amount of pS29 phosphorylation declines modestly during incubation with extract. NE, nuclear extract. Black lines indicate that intervening lanes have been spliced out. (C) HU affects PALB2 binding to RPA. The approach was similar to that used in B, with the exception that both nonperturbed and HU-treated extracts were tested. (D) Quantitation of PALB2 binding to nonphosphorylated (RPA) or phosphorylated (pRPA) RPA. rel. amt, relative amount. The data are expressed as means ± SD.

Figure 7.

Figure 7.

The loss of PALB2 impedes fork recovery after replication stress. (A) Western blot showing efficient knockdown of PALB2 in U2-OS cells. Cells were transfected with a specific siRNA against PALB2 or negative (Neg) control siRNA. (B) Diagram showing the fork labeling procedure used to examine the effect of knockdown of PALB2 on fork movement. (C) PALB2 knockdown causes significant defects in the recovery from stress. (D) Diagram of the fork labeling procedure used to examine the effect of PALB2 on HU-induced replication stress and recovery in WT- and PALB2-rescued PALB2-null EUFA1341 cells. (E) Recovery of DNA synthesis after stress is stimulated by PALB2. Note that the presence of PALB2 did not affect replication fork rates during replication stress. **, P < 0.01, relative to fork rates determined in control cells. Error bars indicate SEMs.

Figure 8.

Figure 8.

Cells with defective RPA phosphorylation or lacking PALB2 show increased micronuclei formation as a result of stress. (A) Scheme used to assess DNA damage in U2-OS cells expressing an RPA phosphorylation mutant on recovery from HU treatment. Cells were either exposed to 5 mM HU for 4 h and then allowed to recover for 5 h (i), treated with the PARP inhibitor veliparib (PARPi) for 25 h (ii), or treated overnight with veliparib followed by 4-h HU treatment and 5-h recovery phases (iii), also in the presence of veliparib. (B) TUNEL staining in U2-OS cells in which endogenous RPA2 was replaced with WT-RPA2 or Cdk_A-RPA2. Cells are shown after recovery from HU treatment or after recovery from HU treatment in the presence of veliparib (as indicated). Cells were stained with DAPI (blue) and processed using a TUNEL assay involving fluorescein-12-dUTP incorporation (green). Micronuclei are evident as green bodies found at the outer edges of nuclei. To increase the number of cells shown, the image of cells expressing WT-RPA2 and recovering from HU/PARP inhibitor treatment is a composite. Below the main images is an enlarged image showing micronuclei formation observed in HU/PARP inhibitor–treated cells replaced with Cdk_A-RPA2. Bars: (main images) 40 µm; (enlarged images) 15 µm. (C) Mutation of RPA2 phosphorylation sites increases micronuclei formation after replication stress. The TUNEL-positive micronuclei signal of U2-OS cells replaced with WT-, Cdk_A-, or PIKK_A-RPA2 showing TUNEL-positive micronuclei formation in unperturbed cells or cells treated with veliparib alone for 25 h, 5 h after a 4-h HU treatment, and 5 h after 4-h HU treatment in the presence of veliparib are shown. Mutation of Cdk sites, and to a lesser extent PIKK sites, on RPA2 causes an increase in micronuclei formation after HU/PARP inhibitor treatment. **, P < 0.01, relative to micronuclei levels determined in WT-RPA cells for the similar condition. (D) PALB2 protects against micronuclei formation during recovery from replication stress. The TUNEL-positive micronuclei signals of U2-OS cells treated with siRNA to knockdown PALB2 or control siRNA (Neg., negative) and treated with HU and veliparib, as indicated, are shown. The data are expressed as means ± SD.

Figure 9.

Figure 9.

Model indicating the protective effect of RPA phosphorylation and PALB2 on fork stability during replication stress. (A) An unperturbed replication fork has nonphosphorylated RPA bound to the lagging strand template, with this RPA turned over rapidly during fork movement. (B) Replication fork stalling and consequent helicase-DNA polymerase uncoupling (not depicted) cause the generation of persistent ssDNA on both the leading and lagging strand templates that is stably bound by RPA. This RPA becomes phosphorylated on RPA2 by ATR and Cdk. (C) Phosphorylated RPA recruits PALB2 (and likely BRCA2) to the fork, stabilizing the stalled fork complex. (D) Alleviation of stress conditions leads to RPA dephosphorylation, reducing PALB2 binding to the RPA–-ssDNA complex. The previous binding of PALB2 to the replisome facilitates a rapid revival of fork movement. (E) Deregulated RPA phosphorylation or loss of PALB2 causes reduced protection of the RPA–ssDNA complex. (F) This defective protection is evinced upon the return to nonstress conditions because recovery of fork movement is diminished, and forks are more prone to collapse. P, phosphorylation.

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