1. Chromosomes and Gene Expression
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Translesion polymerase kappa-dependent DNA synthesis underlies replication fork recovery

  1. Peter Tonzi
  2. Yandong Yin
  3. Chelsea Wei Ting Lee
  4. Eli Rothenberg
  5. Tony T Huang  Is a corresponding author
  1. New York University School of Medicine, United States
Research Article
Cite this article as: eLife 2018;7:e41426 doi: 10.7554/eLife.41426
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
PolK is required for replication fork restart due to nucleotide deprivation.

(A) Schematic for measuring replication fork restart by DNA fiber analysis. Quantification of fork restart efficiency (% stalled forks) in HU-treated (2 mM) RPE-1 cells using two independent siRNAs against individual TLS Pols as indicated. Representative images of the DNA fiber tracts are shown. (B) Quantification of fork restart efficiency in RPE-1 cells comparing different HU (2 mM) treatment time-points in the presence or absence of PolK siRNA knockdown. (C) Quantification of fork restart efficiency in HU-treated RPE-1 cells with either a wash step with fresh media or with no wash (HU still present) supplemented with 250 μM deoxynucleosides (dNs) for recovery. (D) Western blot analysis of RPE-1 cells treated with 2 mM HU for 4 hr followed by either a wash step with fresh media or no wash (HU still present) supplemented with 250 μM deoxynucleosides (dNs) or 250 μM ribonucleosides (rNs) for 30 or 60 min chase. (E) Quantification of fork restart efficiency comparing fork-stalling agents, HU (2 mM) or APH (5 μM), in the presence or absence of PolK siRNA knockdown. (F) Western blot analysis of RPE-1 cells treated with either HU (2 mM), APH (5 μM), or Gemcitabine (Gem, 1 μM) for 4 hr, followed by a wash step and recovery in fresh media for 2 hr. Data for % stalled forks are represented by mean ± s.d. of three independent experiments and p-values calculated using t-test with Welch’s correction. n.s. = no significance, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

https://doi.org/10.7554/eLife.41426.002
Figure 1—figure supplement 1
siRNA knockdown efficiencies and complementation of CRISPR 293T sgPolK clonal cells.
https://doi.org/10.7554/eLife.41426.003
Figure 1—figure supplement 2
Gemcitabine-induced stalled forks are not amenable for fork restart assays.
https://doi.org/10.7554/eLife.41426.004
PolK functions in concert with the FA pathway to promote replication fork restart.

(A) Quantification of fork restart efficiency in FANCD2-deficient patient cells (PD20) complemented with either vector only, FANCD2 WT, or K561R mutant in the presence or absence of PolK siRNA and treated as indicated. PD20 (vector only) cells were treated with 300 nM Chk1i (AZD7762) throughout the duration of HU and CldU time points as a positive control for the detection of elevated fork-stalling events. Western blot analysis showing siRNA knockdown efficiency in PD20 cells. (B) Quantification of fork restart efficiency in 293T CRISPR PolK (sgPolK) cells complemented with either empty vector or GFP-PolK WT in the presence or absence of FANCD2 siRNA and treated as indicated. CldU (red) tract length measurements of restarted forks determine the varying degree of individual fork restart events. Western blot analysis showing expression and siRNA knockdown efficiency in sgPolK 293 T cells. Data for % stalled forks are represented by mean ± s.d. of three independent experiments and p-values calculated using t-test with Welch’s correction. Data for tract length measurements are plotted from three independent experiments with mean ± s.e.m. and p-values calculated using Mann-Whitney t-test. n.s. = no significance, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

https://doi.org/10.7554/eLife.41426.005
Figure 3 with 2 supplements
PolK interacts with K48-linked polyubiquitinated PCNA via its UBZ domains in a HU-dependent manner.

(A) Western blot analysis of RPE-1 cells treated with the indicated siRNAs and HU (2 mM) time-points. (B) Western blot analysis of 293T sgPolK cells complemented with either empty vector or GFP-PolK WT and pulsed with HU (2 mM) for 4 hr before wash step and recovery for the indicated time-points. (C) Schematic diagram showing domains of PolK. Formaldehyde-induced crosslinking of 293T sgPolK cells treated with HU (2 mM) for 4 hr as indicated. Extracts from cells complemented with either empty vector, GFP-PolK WT or a double ubiquitin-binding domain mutant (UBZ) of GFP-PolK were then subjected to anti-GFP pulldown, followed by Western blot analysis with the indicated antibodies. (D) Ubiquitin chain restriction digest analysis using similarly treated and immunoprecipitated (IP) samples as in (C) to enrich for polyubiquitinated PCNA that is bound by GFP-PolK and induced by HU. Samples on beads were then incubated with 900 ng of indicated recombinant DUBs for 1 hr at 37°C prior to Western blot analysis with the indicated antibodies (upper and lower panels). SARS PLpro catalytic mutant (C112A) was used for negative control as indicated (lower panel). (E) Quantification of fork restart efficiency in 293T sgPolK cells complemented with either empty vector, GFP-PolK WT, Catalytic-Dead (CD), or ubiquitin-binding mutant (UBZ). CldU (red) tract length measurements of restarted forks were determined for WT and the different PolK mutants. (F) Quantification of fork restart efficiency in U2OS cells treated with PCNA siRNA and complemented with siRNA-resistant HA-tagged PCNA-WT or ubiquitin site mutant HA-PCNA K164R. Western blot analysis showing exogenously expressed siRNA-resistant HA-PCNA in U2OS cells. Data for % stalled forks are represented by mean ± s.d. of three independent experiments and p-values calculated using t-test with Welch’s correction. Data for tract measurements are plotted from three independent experiments with mean ± s.e.m. and p-values calculated using Mann-Whitney t-test. n.s. = no significance, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

https://doi.org/10.7554/eLife.41426.006
Figure 3—figure supplement 1
PolK interacts with FANCD2 and RPA independently of its UBZ domain.
https://doi.org/10.7554/eLife.41426.007
Figure 3—figure supplement 2
HU-dependent PCNA polyubiquitination is susceptible to an in vitro K48-specific polyUb DUB cleavage reaction.
https://doi.org/10.7554/eLife.41426.008
Figure 4 with 1 supplement
PolK prevents MRE11-dependent nascent DNA degradation.

(A) Schematic for measuring nascent DNA degradation (shortened CldU-labeled tracts) by DNA fiber analysis (A–C,E). Quantification of nascent DNA degradation (changes in CldU tract lengths) in 293 T cells treated with the indicated siRNAs. (B) Quantification of nascent DNA degradation in 293T sgPolK cells that were complemented with either empty vector, GFP-PolK WT or the indicated GFP-PolK mutants. (C) Quantification of nascent DNA degradation in 293T sgPolK cells complemented with either empty vector or GFP-PolK WT were treated with or without Mre11 inhibitor, Mirin (50 μm), in the presence of HU as indicated. (D) Quantification of fork restart efficiency in RPE-1 cells with the indicated siRNAs and treated with or without Mirin (50 μM) in the presence of HU (2 mM) as indicated. (E) Quantification of nascent DNA degradation in parental 293T or 293T sgPolK cells treated with the indicated siRNAs. (F) Quantification of fork restart efficiency in parental 293T or 293T sgPolK cells treated with the indicated siRNAs. Data for % stalled forks are represented by mean ± s.d. of three independent experiments and p-values calculated using t-test with Welch’s correction. Data for tract length measurements are plotted from three independent experiments with mean ± s.e.m. and p-values calculated using Mann-Whitney t-test. n.s. = no significance, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

https://doi.org/10.7554/eLife.41426.009
Figure 4—figure supplement 1
RAD51 and BRCA2 depletion in RPE-1 cells has no effect on replication fork restart after HU treatment.
https://doi.org/10.7554/eLife.41426.010
Figure 5 with 3 supplements
PolK-dependent DNA synthesis in the presence of HU.

(A) Schematic for measuring replication fork speed (CldU tract length) under HU treatment for 4 hr. To ensure that the CldU-labeled DNA was under constant high dose HU treatment, cells were pre-treated with 2 mM HU for 40 min prior to the addition of CldU in the presence of HU for 4 hr. Quantification of fork speed in RPE-1 cells treated with the indicated siRNAs. (B) Schematic of a modified iPOND assay to measure proteins associated with the replisome under nucleotide starvation conditions. Similar to (A), 293 T cells were either left untreated or pretreated with HU (2 mM) for 40 min prior to the addition of EdU (10 μM) in the presence of HU for 4 hr. Cells were then either collected immediately (EdU samples) or chased with Thymidine (10 μM) for 1 hr (EdU + chase) in the presence or absence of HU. Samples without HU were treated for only 10 min with EdU or chased with Thymidine for 1 hr. Western blot analysis showing the biotin-streptavidin pulldown after click-reaction in parental 293 T cells and probed with the indicated antibodies. (C) Schematic for measuring EdU incorporation intensity by direct fluorescence measurements in HU-treated cells. U2OS sgPolK cells were complemented with either empty vector, GFP-PolK WT, GFP-PolK mutant constructs, or different Y-family TLS Pols, GFP-Pol eta or GFP-Pol iota. Cells were pretreated with HU (2 mM) for 1 hr, prior to the addition of EdU (10 μM) in the presence of HU for 4 hr. Mean EdU intensity per nucleus measured by ImageJ were plotted from three independent experiments. (D) Single-molecule localization imaging of EdU signal distribution per foci or nuclei. RPE-1 cells were treated with the indicated siRNAs prior to pretreatment with HU (2 mM) for 1 hr, followed by the addition of EdU in the presence of HU for 4 hr. Representative super-resolution images of nuclei with EdU signal in magenta are shown. Quantification of EdU foci counts per nuclei and amount of EdU counts per foci are plotted from three independent experiments. (E) U2OS sgPolK cells complemented with either GFP-PolK WT or GFP-PolK CD were pulse-labeled with EdU and treated with HU (2 mM) or not (NT). Treatment conditions and quantification of EdU foci per nuclei by super-resolution imaging techniques were done as in (D). (F) Quantification of fork restart efficiency in RPE-1 cells treated with the indicated siRNAs whereby ‘restarted’ forks are measured as previously elongating forks (IdU tracts) that become converted to CldU tracts in the presence of HU (2 mM). Cells were pretreated with HU for 40 min prior to the addition of CldU for 4 hr to ensure that CldU pulse-labeled cells were already under constant high-dose HU treatment. (G) Quantification of fork restart efficiency in RPE-1 cells treated with the indicated siRNAs whereby IdU pulse-labeled forks under constant HU treatment are measured to determine whether they can be ‘restarted’ after HU wash off (CldU pulse-label). Data for % stalled forks and quantification of EdU foci counts by single-molecule localization imaging are represented by mean ± s.d. of three independent experiments and p-values calculated using t-test with Welch’s correction and indicated above the plots. Data for tract length measurements are plotted from three independent experiments with mean ± s.e.m. and p-values calculated using Mann-Whitney t-test. n.s. = no significance, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

https://doi.org/10.7554/eLife.41426.011
Figure 5—source data 1

EdU intensity per nucleus.

https://doi.org/10.7554/eLife.41426.015
Figure 5—figure supplement 1
Mirin treatment does not affect replication fork speed in either PolK- or FANCD2-depleted RPE-1 cells.
https://doi.org/10.7554/eLife.41426.012
Figure 5—figure supplement 2
PolK-dependent DNA synthesis under HU is unaffected by DRB treatment.
https://doi.org/10.7554/eLife.41426.013
Figure 5—figure supplement 3
Single-molecule localization image of EdU signal, PCNA, and GFP-PolK in U2OS cells.
https://doi.org/10.7554/eLife.41426.014
Figure 6 with 1 supplement
Loss of PolK leads to a p53-dependent cell cycle delay and 53BP1 nuclear body accumulation following HU pulse treatment.

(A) Schematic for measuring cell cycle progression after recovery from HU pulse treatment (A–C). RPE-1 cells treated with the indicated siRNAs were initially pulsed-labeled with EdU (10 μM) for 1 hr to label untreated S-phase cells, followed by a wash step, an HU (2 mM) pulse treatment for 4 hr, another wash step, and recovery (chase) with fresh media for the indicated time. Recovery of EdU-positive, HU pulse-treated cells were tracked by FACS analysis and the proportion of cells in different cell cycle phases were determined by DAPI DNA content (FlowJo). Data represented from three independent experiments with mean ± s.d (A). (B) RPE-1 cells treated with the indicated siRNAs were treated with HU (4 hr), followed by a wash step, and chase for 24 hr with fresh media. Cells were then fixed and stained for SA-β-Gal activity. Data represented from three independent experiments with mean ± s.d., p-value calculated using t-test with Welch’s correction. (C) RPE-1 cells treated with the indicated siRNAs were treated with HU (4 hr), followed by a wash step, and chase for 18 hr with fresh media. Cells were then fixed and co-stained for Cyclin A and 53BP1. Only Cyclin A-negative RPE-1 cells (G1 phase) were quantified for 53BP1 nuclear bodies. Data represented from three independent experiments with a minimum of 300 Cyclin A-negative cells per experiment; mean ± s.d. was plotted and p-value calculated using t-test with Welch’s correction. (D) A model depicting how PolK promotes replication stress recovery and genome stability in an FA pathway-dependent manner in response to conditions of nucleotide starvation. n.s. = no significance, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

https://doi.org/10.7554/eLife.41426.016
Figure 6—figure supplement 1
Mirin treatment does not rescue genome instability in HU pulse-treated PolK- or FANCD2-deficient cells.
https://doi.org/10.7554/eLife.41426.017
Author response image 1
Showing expression of WT vs Catalytic-dead (CD) Pol kappa in Pol kappa sgRNA KO cells.

Even in the absence of HU (2mM), the CD mutant has elevated Chk1 and RPA32 phosphorylation. This is in contrast to the Bétous et al. (2013) study showing that Pol kappa is required for Chk1 activation upon HU treatment.

https://doi.org/10.7554/eLife.41426.021

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional
information
Cell line
(Homo sapiens)
female
hTERT RPE-1
(RPE)
ATCCCRL-4000
Cell line
(Homo sapiens)
female
U-2 OS (U2OS)ATCCHTB-96
Cell line
(Homo sapiens) female
U2OS PolK KO (U2OS sgPolK
Clone #1)
This PaperCRISPR-Cas9 generated using pX330 construct and guide RNAs listed in Materials and methods.
Cell line (Homo sapiens)293TATCCCRL-3216
Cell line (Homo sapiens)293T PolK KO #1 (sgPolK Clone #1)This PaperCRISPR-Cas9 generated using pX330 construct and guide RNAs listed in Materials and methods.
Cell line (Homo sapiens)293T PolK KO #2 (sgPolK Clone #2)This PaperCRISPR-Cas9 generated using pX330 construct and guide RNAs listed in Materials and methods.
Cell line (Homo sapiens)PD20 VectorPMID: 11239454Garcia-Higuera et al., 2001
Cell line
(Homo sapiens)
PD20 FANCD2 WTPMID: 11239454Garcia-Higuera et al., 2001
Cell line
(Homo sapiens)
PD20 FANCD2
K561R
PMID: 11239454Garcia-Higuera et al., 2001
Transfected construct (Homo sapiens)pCDNA3.1 HA-
PCNA WT
PMID: 22157819Dungrawala and Cortez, 2015
Transfected construct
(Homo sapiens)
pCDNA3.1 HA-
PCNA K164R
PMID: 22157819Dungrawala and Cortez, 2015
Recombinant DNA reagenteGFP-C1ClonTech
Transfected construct (Homo sapiens)eGFP-C1-PolK WTPMID: 22157819Dungrawala and Cortez, 2015
Transfected construct (Homo sapiens)eGFP-C1-PolK CDThis PaperSite directed mutagenesis using primers listed in Materials and methods. D198A/E199A
Transfected construct (Homo sapiens)eGFP-C1-PolK UBZThis PaperSite directed mutagenesis using primers
listed in Materials and methods. D644A/D799A
Transfected construct (Homo sapiens)eGFP-C1-PolH WT
Dungrawala and Cortez, 2015
Dungrawala and Cortez, 2015 PMID: 22157819
Transfected construct (Homo sapiens)eGFP-C1-PolI WTDungrawala et al., 2015 PMID: 22157819
Recombinant DNA reagentpX330Addgene#42230
AntibodyMouse anti-IdUBD BioscienceCat. #: 347580IF (1:200)
AntibodyRat anti-CldUAbcamCat. #: ab6326IF (1:100)
AntibodyMouse anti-PCNAAbcamCat. #: ab29WB (1:5000)
AntibodyRabbit anti-PCNA-UbCell SignalingCat. #: D5C7PWB (1:1000)
AntibodyMouse anti-
FANCD2
Santa CruzCat. #: sc-20022WB (1:500)
AntibodyRabbit anti-
FANCD2
Novus BiologicalCat. #: NB100-182WB (1:5000)
AntibodyRabbit anti-pCHK1 S345Cell SignalingCat. #: 133D3WB (1:5000)
AntibodyGoat
anti-CHK1
AbcamCat. #: ab2845WB (1:5000)
AntibodyMouse
anti-gH2AX
EMD MilliporeCat. #: 05–636WB (1:5000)
AntibodyMouse
anti-GFP
Santa CruzCat. #: sc-9996IF (1:200), WB
(1:5000)
AntibodyMouse anti-Cyclin
A2
CalBiochemclone E23IF (1:100)
AntibodyRabbit
anti-53BP1
AbcamCat. #: ab21083IF (1:200)
AntibodyMouse
anti-POLK
Santa CruzCat. #: sc-166667WB (1:1000)
AntibodyMouse
anti-POLH
Santa CruzCat. #: sc-17770WB (1:1000)
AntibodyRabbit
anti-POLI
BethylCat. #: A301-304AWB (1:5000)
AntibodyRabbit
anti-MCM2
BethylCat. #: A300-094AWB (1:5000)
AntibodyRabbit
anti-MCM5
BethylCat. #: A300-195AWB (1:5000)
AntibodyRabbit anti-
Histone H3
AbcamCat. #: ab1791WB (1:5000)
AntibodyRabbit
anti-REV7
AbcamCat. #: ab180579WB (1:5000)
AntibodyRabbit anti-pRPA32 S33BethylCat. #: A300-246AWB (1:5000)
AntibodyRabbit anti-pRPA32 S4/S8BethylCat. #: A700-009WB (1:5000)
AntibodyRabbit
anti-RPA32
BethylCat. #: A300-244AWB (1:5000)
AntibodyMouse
anti-BRCA2
CalBiochemclone 2BWB (1:1000)
AntibodyMouse
anti-HA
BioLegendCat. #: 901502WB (1:5000)
AntibodyGoat
anti-HisTag
BethylCat. #: A190-113AWB (1:5000)
AntibodyRabbit
anti-REV1
Santa CruzCat. #: sc-48806WB (1:1000)
AntibodyRabbit
anti-FANCI
BethylCat. #: A301-254AWB (1:5000)
AntibodyRabbit
anti-MCM4
BethylCat. #: A300-193AWB (1:5000)
AntibodyRabbit
anti-MCM3
BethylCat. #: A300-192AWB (1:5000)
AntibodyRabbit
anti-Rad51
AbcamCat. #: ab63801WB (1:5000)
AntibodyRabbit anti-pRPB1 CTD S2Cell SignalingCat. #: E1Z3GWB (1:1000)
AntibodyMouse
anti-RPB1 CTD
Cell SignalingCat. #: 2629WB (1:1000)
AntibodyRabbit
anti-pCHK2 T68
Cell SignalingCat. #: C13C1WB (1:5000)
Recombinant DNA reagentFugene 6 Transfection reagentPromegaE2692
Recombinant DNA reagentLipofectamine RNAiMAXInvitrogenCat #: 13778150
Sequence-
based reagent
CRISPR guide
RNAs (sgPolK#1,#2)
This PaperSee Materials and methods
Sequence-
based reagent
siRNAsThis PaperSee Materials and methods
Sequence-
based reagent
Mutagenesis
primers
This PaperSee Materials and methods
Peptide,
recombinant
protein
Tri-Ubiquitin chains (K48-linked)Boston BiochemUC-215B
Peptide,
recombinant
protein
Tri-Ubiquitin chains (K63-linked)Boston BiochemUC-315B
Peptide,
recombinant
protein
USP2 Catalytic Domain (CD)Boston BiochemE-504
Peptide,
recombinant
protein
AMSHBoston BiochemE-548B
Peptide,
recombinant
protein
SARS PLProBoston BiochemE-610
Commercial
assay or kit
Click-it EdU Imaging KitInvitrogenC10339
Commercial
assay or kit
Click-it EdU Flow Cytometry Assay KitInvitrogenC10646
Commercial
assay or kit
QuikChange XL Mutagenesis KitAgilentCat #: 200517
Commercial
assay or kit
TOPO-TA Cloning Kit for SequencingInvitrogenCat #: 450071
Chemical
compound, drug
Hydroxyurea (HU)SigmaH8627
Chemical
compound, drug
Aphidicolin (APH)SigmaA4487
Chemical
compound, drug
Gemcitabine (Gem)SigmaG6423
Chemical
compound, drug
MirinSigmaM9948
Chemical
compound, drug
AZD7762 (CHK1 inhibitor)SigmaSML0350
Chemical
compound, drug
1-b-D-ribofuranoside (DRB)SigmaD1916
Chemical
compound, drug
Flavopiridol (FVP)SigmaF3055
Chemical
compound, drug
Formaldehyde 37% w/vVWRM134
Chemical
compound, drug
GlycineFischer ScientificBP381
Chemical
compound, drug
5'-Iodo-2'-deoxyuridine (IdU)SigmaI7125
Chemical compound, drug5'-chloro-2'-deoxyuridine (CldU)SigmaC6891
Chemical
compound, drug
5'-ethynyl-2-deoxyuridine (EdU)SigmaCat #: 900584
Chemical
compound, drug
Thymidine (dT)SigmaT1895
Chemical
compound, drug
2'-deoxycytidine
HCl (dC)
SigmaD0776
Chemical
compound, drug
2'-deoxyadenosine
(dA)
SigmaD8668
Chemical
compound, drug
2'-deoxyguanosine
(dG)
SigmaD0901
Chemical
compound, drug
Cytidine (rC)SigmaC4654
Chemical
compound, drug
Adenosine (rA)SigmaA4036
Chemical
compound, drug
Guanosine (rG)SigmaG6264
Chemical
compound, drug
Uridine (rU)SigmaU3003
Chemical
compound, drug
Anti-GFP mAb agarose beadsMBLCat #: D153-8
Chemical
compound, drug
Dynabeads Myone Streptavidin T1ThermoFisher ScientificCat #: 65601
Chemical
compound, drug
Biotin AzideThermoFisher ScientificCat #: B10184
Chemical
compound, drug
Protein G beads agaroseThermoFisher
Scientific
Cat #: 20399
Chemical
compound, drug
SYPRO Ruby Protein Gel StainThermoFisher ScientificCat #: S12000
Chemical
compound, drug
cOmplete Mini Protease Inhibitor CocktailSigmaCat #: 11836170001 (Roche)
Software,
algorithm
GraphPad Prism(https://graphpad.com)RRID:SCR_015807
Software,
algorithm
ImageJ(https://imagej.nih.gov/ij/)RRID:SCR_003070
Software,
algorithm
FlowJo(https://www.flowjo.com/)RRID:SCR_008520

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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