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, 14 (12), 1104-12

hPrimpol1/CCDC111 Is a Human DNA Primase-Polymerase Required for the Maintenance of Genome Integrity

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hPrimpol1/CCDC111 Is a Human DNA Primase-Polymerase Required for the Maintenance of Genome Integrity

Li Wan et al. EMBO Rep.

Abstract

Prim-pol is a recently identified DNA primase-polymerase belonging to the archaeao-eukaryotic primase (AEP) superfamily. Here, we characterize a previously unrecognized prim-pol in human cells, which we designate hPrimpol1 (human primase-polymerase 1). hPrimpol1 possesses primase and DNA polymerase activities in vitro, interacts directly with RPA1 and is recruited to sites of DNA damage and stalled replication forks in an RPA1-dependent manner. Cells depleted of hPrimpol1 display increased spontaneous DNA damage and defects in the restart of stalled replication forks. Both RPA1 binding and the primase activity of hPrimpol1 are required for its cellular function during DNA replication. Our results indicate that hPrimpol1 is a novel factor involved in the response to DNA replication stress.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Identification of hPrimpol1/CCDC111 as a novel RPA-binding partner. (AD) 293T cells stably expressing SFB-tagged-RPA or hPrimpol1/CCDC111 were used for TAP of protein complexes specifically from chromatin fractions. Tables are summaries of proteins identified by mass spectrometry analysis. Letters in bold indicate the bait proteins. (E) hPrimpol1 interacts with RPA1. 293T cells were transfected with indicated plasmids. Cell lysates were immunoprecipitated with anti-Flag antibody and western blot analysis was performed as indicated. (F,G) Association of endogenous hPrimpol1 with the RPA complex in HeLa cells was performed by co-immunoprecipitation using anti-RPA2 or anti-hPrimpol1 antibody. HeLa cells were lysed in the presence of benzonase, cell lysates were then incubated with protein A agarose beads conjugated with indicated antibodies and western blot analysis was carried out as indicated. (H) Direct binding between recombinant GST-tagged hPrimpol1 and MBP-tagged-RPA. Upper panel: hPrimpol1 was detected by immunoblotting. Lower panel: Purified proteins visualized by Coomassie staining. (I) hPrimpol1 colocalizes with RPA2. SFB-tagged hPrimpol1 was expressed in 293T cells. Foci assembled by this fusion protein and by RPA2 following exposure to HU (2 mM) for 16 h or IR (10 Gy) followed by recovery for 3 h were detected by immunofluorescence using anti-Flag and anti-RPA2 antibodies, respectively. A merged image shows colocalization. Scale bar, 10 μm. GST, glutathione S-transferase; hPrimpol1 human primase-polymerase 1; MBP, maltose binding protein; RPA, replication protein A; TAP, tandem affinity purification.
Figure 2
Figure 2
The conserved C terminus of hPrimpol1 is required for RPA1 binding and its foci formation. (A) Schematic representation of wild-type and deletion mutants of hPrimpol1 used in this study. (B) The 80 amino acids at the C terminus of hPrimpol1 are required for RPA1 binding. Co-immunoprecipitation experiments were carried out as indicated. (C) Alignment of C-terminal sequences of hPrimpol1 from different species. (D) The conserved C terminus of hPrimpol1 is responsible for its foci formation. Immunostaining experiments were performed 16 h after HU treatment using indicated antibodies. Scale bar, 10 μm. (E) The conserved C terminus of hPrimpol1 is sufficient for its foci formation. 293T cells were transfected with SFB-tagged T5 mutant of hPrimpol1. Immunostaining experiments were performed using indicated antibodies. Scale bar, 10 μm. (F) The C-terminal fragment (T5) of hPrimpol1 is sufficient for RPA1 binding. Co-immunoprecipitation experiments were carried out as indicated. hPrimpol1 human primase-polymerase 1; HU, hydroxyurea; RPA, replication protein A.
Figure 3
Figure 3
hPrimpol1 possesses primase and DNA polymerase activities. (A) SDS–PAGE profile of purified wild-type and mutants of hPrimpol1. (B) hPrimpol1 is a bona fide primase. Wild type and mutants of hPrimpol1 were incubated with ssDNA template (T20GTCT20) and NTPs containing [α-32P]-ATP as substrates. Reaction products were separated by 8 M Urea-SDS–PAGE and detected by autoradiography. (C) hPrimpol1 displays DNA polymerase activity. The ability of wild-type and mutant proteins to synthesize DNA was analysed by classical tritium incorporation assay. Incorporation of [3H] dTTP into poly(dA)500/oligo(dT)18 was quantified by scintillation counting. Error bars are s.d.; n=3. (D) Both wide-type and the D5 mutant of hPrimpol1 can elongate the primers synthesized by its own primase activity efficiently. Reactions were carried out as standard primase assays in Fig. 3B except that 100 μM dNTPs were added together with 100 μM NTPs containing [α-32P]-ATP. (E) SDS–PAGE profile of purified D5 mutant of hPrimpol1. hPrimpol1 human primase-polymerase 1; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; ssDNA, single-stranded DNA.
Figure 4
Figure 4
hPrimpol1 functions at stalled replication forks. (A,B) Clonogenic survival assays in hPrimpol1-depleted HeLa cells following HU or IR treatment. Error bars are s.d.; n=3. (C,D) γH2AX foci are greatly increased in hPrimpol1-depleted HeLa cells. HeLa cells were infected with lentiviruses carrying non-target control or hPrimpol1 shRNAs. Seventy-two hours later, cells were subjected to immunostaining using indicated antibodies (C). Scale bar, 10 μm. The quantification of foci-positive cells was performed by counting a total of 200 cells per sample (D). Error bars are s.d.; n=3. (E) Quantification of chromosomal aberrations in control and hPrimpol1-depleted HeLa cells. At least 50 cells were counted in each experiment. Error bars are s.d.; n=3; significances of differences by a two-tailed unpaired t-test. (F) Schematic representation of the labelling protocol for DNA fibre analysis of replication forks. (G,H) CIdU tract length distributions from DNA fibres from control and hPrimpol1-depleted HeLa cells in the presence (H) or absence of HU (G). (I) hPrimpol1 is not required for CHK1 activation. HeLa cells were introduced with indicated shRNA and then treated with HU (2 mM) for 2 h. Cells were lysed and immunoblotting was performed using indicated antibodies. hPrimpol1 human primase-polymerase 1; HU, hydroxyurea; shRNA, small hairpin RNA.
Figure 5
Figure 5
The RPA1 binding and the primase activity of hPrimpol1 are required for its cellular functions. (A) shRNA-resistant hPrimpol1 and its indicated mutants were transduced into hPrimpol1-deplected HeLa cells. The indicated proteins were analysed in HeLa cells cultured with doxycycline (Dox) for 24 h. (B,C) hPrimpol1 depletion-induced increase of γH2AX foci was rescued by the expression of wild-type hPrimpol1 but not the D5 and the enzyme-inactivating mutants of hPrimpol1. shRNA-resistant hPrimpol1 and its indicated mutants were transduced into hPrimpol1-depleted HeLa cells. Cells were cultured with Dox for 24 h and then subjected to immunostaining using indicated antibodies (B). Scale bar, 10 μm. The quantification of foci-positive cells was performed by counting a total of 200 cells per sample (C). Error bars are s.d.; n=3. (D) Both RPA1 binding and the primase activity of hPrimpol1 are required for restoring cellular resistance to HU. Error bars are s.d.; n=3. (E,F) Defects in fork restart in hPrimpol1-deplected HeLa cells were rescued by wild-type hPrimpol1 but not by its mutants. Quantification of CIdU track lengths was shown (E). Images of DNA fibres isolated from cells treated with hPrimpol1 shRNA following the expression of shRNA-resistant wild-type, the D5, or the enzyme-inactivating mutants of hPrimpol1 (F). Scale bar, 10 μm. (G) A proposed model of hPrimpol1 function at replication forks. Please refer the text for details. hPrimpol1 human primase-polymerase 1; HU, hydroxyurea; shRNA, small hairpin RNA.

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