Fig 1.
The structural importance of PCNA Ser46, and Leu47 residues in a central loop and hydrophobic core formation.
(A) (Upper) Diagram indicates all the available coding exons (gray bar) in the pcna gene and the targeted exon region is indicated with red arrowheads. (Bottom) DNA sequence (112–150) of zebrafish pcna and the sequences of sgRNA target site between WT and pcna in-frame mutant (ΔSer46-Leu47, ΔSL47) in red. Putative translated amino acid sequences are indicated below the DNA sequences. Hyphens indicate deleted sequence of pcna ΔSL47 mutant. (B) Amino acid sequence and the secondary structure of human PCNA (P12004) are shown. α-helices and β-strands are indicated above the amino acid sequence. Ser46 and Leu47 residues are colored in blue. (C) Human PCNA structure containing central loop and inter-domain connector loop (IDCL) regions marked with red. (D) Conserved residues in PCNA. (E) Predicted monomer structures of PCNAWT (Green) or PCNAΔSL47 (Cyan) in both human and zebrafish. The red circle indicates the central loop region. (F) Hydrophobicity of PCNAWT and PCNAΔSL47 monomers around PCNA central loop region. The closer the surface color is to red, the more hydrophobic it is. Hydrophobicity is analyzed in both human and zebrafish. The blue circle indicates the Ser46-Leu47 residues. (G-I) Predicted trimerized PCNA structure analyzed with corresponding combinations in human. PCNA WT-WT-WT homotrimer (Gray) are structurally aligned with either WT-WT-ΔSL47 (Green) (G), WT- ΔSL47-ΔSL47 (Cyan) (H), ΔSL47-ΔSL47-ΔSL47 (Yellow) (I). Both the front- and side- views are displayed (Top). The central loop regions are colored as indicated (Bottom).
Fig 2.
Purified PCNAΔSL47 protein forms non-trimeric heterogenous multimers in vitro.
(A) Coomassie-stained SDS-PAGE of purified PCNAWT, PCNAΔSL47, RFC, and TALE proteins. (B) Schematic diagram of PCNA loading assay. 5’-biotinylated DNA substrate was attached to the streptavidin-coated magnetic beads. The free end of DNA was blocked by the TALE protein. PCNA was loaded to the substrate DNA by purified RFC. (C) DNA-loaded PCNA is analyzed by immunoblotting. (D) The indicated amount of purified PCNAWT or PCNAΔSL47 was separated on 10% non-denaturing gel and stained with Coomassie blue. (E) Gel filtration analysis with purified PCNAWT or PCNAΔSL47 mutant was performed and stained with Coomassie blue. (F) Myc- or HA-tagged PCNA constructs were expressed in HEK293T cells and subjected to immunoprecipitation (IP) with anti-Myc affinity beads. Immunoprecipitated proteins were eluted, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies.
Fig 3.
PCNAΔSL47 mutant interacts with RFC subunits but fails to interact with FEN1 and LIG1.
(A-F) Immunoprecipitated proteins are eluted, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. (A-D) After transfection in HEK293T cells as indicated, nuclear extracts were prepared for immunoprecipitation (IP) with an anti-FLAG affinity (A and B) or anti-HA affinity (C), or anti-S affinity bead (D). (C and D) The intensity ratio of either Ub-PCNA and HA (C) or Ub-PCNA and S (D) was quantified and plotted. (E and F) After transfection with either V5-tagged FEN1 or LIG1 in HEK293T cells for 48 h, whole-cell lysates were prepared and incubated with purified PCNA protein for 1 h.
Fig 4.
PCNA Ser46 and Leu47 amino acids are important for genomic stability in cells.
(A and B) HEK293T cells were transfected with a combination of cDNA expressing HA-PCNAWT (A and B) or HA-PCNAΔSL47 (B) and PCNA siRNA targeting 3’UTR for 48 h. Whole-cell extracts were prepared for immunoblotting. (C) After cDNA transfection as indicated, cells were irradiated with 50 J/m2 UV and recovered for 6 h or treated for 6 h with 30 μM USP1 inhibitor (ML323). Whole-cell extracts were isolated and immunoblotted. (D) HEK293T cells were transfected with cDNA expressing HA-PCNAWT or HA-PCNAΔSL47 for 48 h and chromatin-bound fractions were prepared for immunoblotting. The intensity ratio of Ub-PCNA and PCNA was quantified and plotted. (E and F) HeLa cells were transfected with cDNA expressing HA-tagged PCNAWT or PCNAΔSL47 for 48 h, and immunostained with the anti-S9.6 antibody. (E) Representative images of S9.6 immunostaining. (F) The sum intensity of S9.6 foci was quantified and plotted. (G) After cDNA transfection as indicated in HEK293T cells, cells were irradiated with 50 J/m2 UV and recovered for 6 h. Whole-cell extracts were prepared for immunoblotting. (H) HeLa cells were transfected with cDNA expressing S-tagged PCNAWT or PCNAΔSL47 for 48 h, and immunostained with the anti-γH2AX antibody. 10 μM of etoposide was treated for 5 h as a positive control. The mean intensity of γH2AX staining was quantified and plotted. (I) (Upper) Representative images of alkaline COMET assay. U2OS cells were transfected with indicated cDNAs for 48 h, treated with 0.1 mM H2O2 for 1 h, and collected for an alkaline COMET assay. (Below) The tail moments were quantified and plotted. (J and K) U2OS cells were transfected as indicated for 48 h, and treated with H2O2 for 4 h with MTT reagents (J) or irradiated with UV as indicated (K). Error bars represent the standard deviation of the mean. (J) Cell survival was measured by quantification of MTT formazan. (K) Cells were recovered for 48 h after UV-irradiation, and cells were subjected to CellTiter-Glo® assay. (E, F, H, I, J, and K) Three independent experiments were performed, and one representative result is displayed. The red bar indicates the mean value. Statistical analysis: two-tailed unpaired Student’s t-test, **** p < 0.0001, *** p < 0.001, ** p <0.01, * p < 0.05.
Fig 5.
PCNA Ser46-Leu47 and 41DSSHV45 residues are required for maintaining genomic stability in an epistatic manner.
(A) DNA sequences of PCNAWT and central loop-associated PCNA mutants were aligned. (B) After transfection as indicated in HEK293T for 48 h, nuclear extracts were prepared for immunoprecipitation with an anti-S protein affinity bead for 2 h. Immunoprecipitated proteins were eluted, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. (C) HEK293T cells were transfected as indicated for 48 h, and whole-cell extracts were prepared for immunoblotting. (D, E) The sum intensity of S9.6 foci (D) or the mean intensity of γH2AX staining (E) was quantified and plotted. (E) 10 μM etoposide was treated for 5 h as a positive control. (F) Predicted monomer structures of human PCNA-WT, ΔSL47, D41A, D41A;S42A, and SHV43AAA and double mutant with ΔSL47 as indicated. The magnified region indicates the PCNA central loop. (D, E) Three independent experiments were performed, and one representative result is displayed. The red bar indicates the mean value. Statistical analysis: two-tailed unpaired Student’s t-test, **** p < 0.0001, *** p < 0.001, * p < 0.05, and n. s. = not significant.
Fig 6.
Graphical models of PCNA Ser46 and Leu47 residues are crucial in genomic integrity.
(A) During DNA replication, PCNAWT forms a homotrimer and is loaded onto the lagging strand of DNA by the RFC. After RNA primer synthesis by primase and Pol α at the lagging strand, PCNA and POL δ-meditated DNA synthesis occurs. FEN1 and LIG1 interact with PCNAWT homotrimer and process 5’ overhang flap structures and seal the nick. In addition, when the DNA replication fork encounters DNA lesions, PCNA is ubiquitinated and proceeds with translesion synthesis (TLS). Therefore, normal DNA replication proceeds and ensures genomic integrity. (B) When Ser46-Leu47 residues of PCNA are deleted, the PCNAΔSL47 mutation monomer disrupts the PCNA trimerization in vitro. The central loop formation of the PCNAΔSL47 mutant is predicted to be structurally distorted and its hydrophobicity is expected to decrease. PCNA ubiquitination is defective, and high levels of DNA-RNA hybrids and γH2AX are detected in PCNAΔSL47-expressing cells. Consistently, PCNAΔSL47-expressing cells are sensitive to several DNA-damaging agents. Taken together, PCNA Ser46-Leu47 residues are crucial for genomic integrity.