Figures and data
Influence of downstream nucleosome on Polβ activity in linker DNA region.
(a) Schematic structures of DNA and nucleosome, and electrophoregram showing DNA extension after incubation of gap-DNA or gap-NCP (50 nM) without or with Polβ (5 or 20 nM) and all four dNTPs (100 μM each) for 10 min. (b) Time-dependent extension of DNA upon incubation of 50 nM naked or nucleosomal gapped DNA (gap-DNA, lanes 1-5; gap-NCP, lanes 6-10) with Polβ (20 nM) and four dNTPs (100 μM each). (c) Time-dependent gap-filling reaction catalyzed by Polβ (4 nM) on gap-DNA or gap-NCP (50 nM) in the presence of dTTP (0.5 μM). The extent of substrate conversion is indicated at the bottom of the electropherograms. (d) Strand-displacement synthesis products generated by Polβ (25 nM) after incubation with gap-DNA (50 nM; lanes 1, 2) or gap-NCP (50 nM, lanes 3, 4) and four dNTPs (100 μM each), in the absence or presence of FEN1 (25 nM) for 5 min. Positions of substrate (S) and extension products (+n) in denaturing 20% PAG are indicated on the left and right of gel images.
Formation of Polβ complexes with gapped NCP substrate.
(a) Mass photometry data: molecular mass distribution of species in samples containing gap-NCP227 (5 nM) or its mixtures with Polβ (15 or 25 nM). (b) Calculated molecular masses of gap-NCP227 and its Polβ-associated complexes of different stoichiometries.
EC50 values (nM) for protein complexes with DNA and nucleosomes
Influence of linker histone H1 on Polβ activity.
(a) Electrophoregrams show DNA extension by Polβ via strand-displacement synthesis after incubation of Polβ (50 nM) with four dNTPs (100 μM each), H1 (70 nM) and gap-DNA or gap-NCP (50 nM) for 10 min. Positions of the substrate and products of DNA synthesis in denaturing 20% PAG are indicated on the left and right of gel images. On the right are curves reflecting relative intensities of bands in samples compared (marked with color under the electropherogram). (b) Scheme shows proposed models of DNA extension by Polβ via strand-displacement synthesis on NCP or its complex with histone H1.
Comparison of PARP1 and PARP2-induced effects on Polβ activity.
a, b - Electrophoregrams show DNA extension by Polβ via strand-displacement synthesis after incubation of Polβ (3, 20 or 50 nM) with four dNTPs (100 μM each), PARP1/PARP2 (100 nM) and gap-DNA/gap-NCP (50 nM) for 10 min. c, d - DNA extension by Polβ via strand-displacement synthesis after incubation of Polβ (20 nM) with four dNTPs (100 μM each), PARP1/PARP2 (50, 100 or 200 nM) and gap-DNA/gap-NCP (50 nM). On the right are curves reflecting relative intensities of bands in samples compared (marked with color under the electropherogram). Positions of substrate and products of DNA synthesis in denaturing 20% PAG are indicated on the right of gel images.
Influence of H1 and PARylation on Polβ activity.
(a, b) Electrophoregrams show DNA extension by Polβ via strand-displacement synthesis after incubation of Polβ (50 nM) with four dNTPs (100 μM each), H1 (75 nM), PARP1/PARP2 (100 nM), NAD+ (10 μM) and gap-NCP (50 nM). PARylation was carried out by preliminary incubation of NCP mixtures with PARP1/PARP2 and NAD+, with or without H1, for 30 min; further incubation with Polβ was performed for 10 min. Positions of substrate and products of DNA synthesis in denaturing 20% PAG are indicated on the left of gel images. On the right are curves reflecting relative intensities of bands in samples compared (marked with color under the electropherogram).
PARylation of histone H1 depends on HPF1.
a, b - Autoradiograms show covalent binding of 32P-labelled PAR or MAR to protein targets after incubation of PARP1/PARP2 (500 nM) with [32P]NAD+ (1 μM), H1 (70 nM), HPF1 (1 μM) and gap-NCP (250 nM) for 30 min, before (a) and after (b) subsequent treatment of reaction mixtures with PARG. Positions of ADP-ribosylated proteins and their native forms (and molecular weight markers) in 20% SDS-PAG are indicated on the left and right of gel images. c - Histograms show the amount of PAR attached to PARP1/PARP2, H1 and histones in the distinct samples (the mean ± SD, n=3). d, e - Autoradiograms show covalent binding of 32P-labelled MAR to H1 after incubation of PARP1/PARP2 (500 nM) with [32P]NAD+ (1 μM), H1 (d, 300 nM; e, 500 nM), HPF1 (1 μM) and gap-NCP (250 nM) for 45 min,, and following PARG treatment. Positions of mono-ADP-ribosylated H1 and its native form in 20% SDS-PAG are indicated on the left and right of gel images.
A model of multi-layered regulation of Polβ activity in a nucleosomal context.
The nucleosome core particle acts as a platform that facilitates the coordinated action of BER enzymes at a proximal damage site in linker region. Linker histone H1 and PARP1 suppress DNA synthesis; however, activation and autoPARylation of PARP1 at the lesion alleviates this restriction. PARP2, by inhibiting strand-displacement synthesis, serves as a molecular switch from long-patch (LP) to short-patch (SP) BER.
