Fig. 4: In human 293 T cells and outside of cells, POLD3 directly interacts with histone (H3-H4)2 tetramers. | Cell Death & Differentiation

Fig. 4: In human 293 T cells and outside of cells, POLD3 directly interacts with histone (H3-H4)2 tetramers.

From: Proximal proteomics analysis reveals DNA polymerase δ subunit 3 is a new MCM2 binding partner and promotes parental histones inheritance in mammalian cells

Fig. 4

A Top: The known functional domain of POLD3; Bottom: Western blot analysis showing that POLD3-HA co-immunoprecipitated with histone H3 in 293 T cells. This assay further revealed that POLD3’s C-terminal fragment (amino acids 145-466), but not its-N terminal fragment (amino acids 1-144), interacted with histone H3. B Western blot using Flag-tagged histone H3 in 293 T cells demonstrated co-immunoprecipitation of POLD3, histone H3, and MCM2 from soluble cell extract, confirming their interaction. H2A and H2B were tested to exclude the interaction from nucleosomes. C In vitro interaction analysis using purified full-length strep-tagged POLD3 protein and purified (H3-H4)2 tetramer complex revealed strep-tagged POLD3 directly interacted with histone (H3-H4)2 tetramers, similar to the known parental histone chaperone, POLA1. Proteins and histones were stained with Coomassie Brilliant Blue (CBB). D In vitro interactions using purified full-length strep-tagged POLD3 protein, (H3-H4)2 tetramer complex and H2A-H2B dimer complex revealed that strep-tagged POLD3 did not interact with histone H2A-H2B dimers. Proteins and histones were stained with CBB.

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