Fig 2: The POLE3-POLE4 Complex Is a Bona Fide H2A-H2B Histone Fold Complex(A) Cartoon depicting human POLE3 and POLE4 proteins and their predicted structural motifs. a helices are represented as squared boxes, while histone fold motifs are indicated by brackets.(B) Modeled POLE3-POLE4 dimer with conserved Phe44 and Phe74 in stick format. Consurf was used to color residues by conservation from blue (most conserved) to red (least conserved).(C) In vitro GST pull-downs of the indicated GST-tagged proteins in the presence of His-POLE4 (left) or His-POLE3 (right).(D) Western blot analysis of FLAG IPs from whole-cell extracts of HeLa TRex-expressing empty FLAG, FLAG-POLE3 WT, or F44D mutant.(E) Western blot analysis of FLAG IPs from whole-cell extracts of HeLa TRex-expressing empty FLAG, FLAG-POLE4 WT, or F74D mutant.

Fig 3: The POLE3-POLE4 Complex Interacts In Vitro with H3-H4(A) In vitro GST pull-down of the GST-POLE4/His-POLE3 complex in the presence of H3-H4 at the described NaCl concentrations.(B) In vitro GST pull-down of the indicated GST-tagged proteins in the presence of H3-H4. Experiments were performed in 150 mM NaCl.(C) In vitro GST pull-down of the indicated GST-tagged proteins in the presence of H2A-H2B dimers. Experiments were performed in 150 mM NaCl.(D) Limited trypsin digestion of recombinant POLE3-POLE4 complex in the presence of the indicated trypsin concentrations.(E) Analytical gel filtration of POLE3-POLE4, H3-H4, and POLE3-POLE4-H3-H4 complexes in 300 mM NaCl concentrations.(F) SDS-PAGE analysis of gel filtration fractions from (E).

Fig 4: H/D Exchange Mass Spectrometry and Protein Pull-Downs Identify Interaction Domains between POLE3-POLE4 and H3-H4 Complexes(A) Difference plot of hydrogen/deuterium (H/D) exchange data from POLE3 (upper left), POLE4 (upper right), H3 (lower left), and H4 (lower right). A cartoon depicting the domains of the analyzed proteins is shown on the top of the graph. Experimental error is reported in gray, while different colors represent different D2O incubation times. More exposed and protected regions are located, respectively, on the higher and lower part of the graph. Peptide aa numbers from highly protected or exposed regions of POLE3 and H4 are shown. ±0.5 Dalton difference is indicated with red/blue lines, which represents 98% confidence limit, so that peptides above or below these lines can be considered significantly changed. Difference heatmaps are provided in Figure S3A.(B) Analytical gel filtration of the described protein complexes in 300 mM NaCl concentrations.(C) SDS-PAGE analysis of gel filtration fractions from the described protein complexes.

Fig 5: The POLE3-POLE4 Complex Binds to H3-H4 Dimers and Tetramers and Promotes Tetrasome Formation and Supercoiling In Vitro(A) DSS crosslinking experiments performed in 150 mM NaCl. Lane 4 shows control without crosslinking; lanes 1, 2, and 5 show samples incubated with 1 mM DSS for 30 min at 23°C and resolved by SDS-PAGE and Coomassie staining. Lane 3 was left empty. Two black arrows indicate the molecular weight species identified upon crosslinking of POLE3-POLE4/H3-H4 co-complex. T (tetramers) indicates POLE3-POLE4 binding to H3-H4 tetramers, while D (dimers) indicates POLE3-POLE4 binding to dimers.(B) Western blot analysis of DSS crosslinking experiments performed in 150 mM NaCl. Lane 1 shows control without crosslinking; lane 2 shows sample incubated with 1 mM DSS for 30 min at 23°C. Samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with a-H3, a-H4, a-POLE3, and a-POLE4 antibodies, from left to right.(C) Analytical gel filtration of POLE3-POLE4 complex alone or in combination with H3-H4 or H3(EE)-H4. Experiments were performed in 300 mM NaCl to prevent histone aggregation.(D) SDS-PAGE analysis and Coomassie staining of gel filtration fractions from (C).(E) Tetrasome assembly on linear DNA (Widom 601 sequence) monitored by native PAGE. Lane 2 shows tetrasome preassembled by salt dilution method; lanes 3–5 show linear DNA incubated with the indicated proteins.(F and G) Tetrasome assembly on linear DNA, performed as in (E), with the indicated proteins.(H) Plasmid supercoiling assay resolved by native agarose gel electrophoresis. Lane 1 shows supercoiled control phix174 RF1 DNA; lane 2, phix174 RF1 DNA relaxed by TOPO I; and lanes 3–7, phix174 RF1 DNA incubated, in the presence of TOPO I, with histones and increasing concentrations of POLE3-POLE4.(I) Plasmid supercoiling assay performed as described in (H) using increasing concentrations of POLE3?C-POLE4 or POLE3-POLE4.