Rotational dynamics of DNA on the nucleosome surface markedly impact accessibility to a DNA repair enzyme

Abstract

Histones play a crucial role in the organization of DNA in the nucleus, but their presence can prevent interactions with DNA binding proteins responsible for repair of DNA damage. Uracil is an abundant mutagenic lesion recognized by uracil DNA glycosylase (UDG) in the first step of base excision repair (BER). In nucleosome core particles (NCPs), we find substantial differences in UDG-directed cleavage at uracils rotationally positioned toward (U-In) or away from (U-Out) the histone core, or midway between these orientations (U-Mid). Whereas U-Out NCPs show a cleavage rate just below that of naked DNA, U-In and U-Mid NCPs have markedly slower rates of cleavage. Crosslinking of U-In DNA to histones in NCPs yields a greater reduction in cleavage rate but, surprisingly, yields a higher rate of cleavage in U-Out NCPs compared with uncrosslinked NCPs. Moreover, the next enzyme in BER, APE1, stimulates the activity of human UDG in U-Out NCPs, suggesting these enzymes interact on the surface of histones in orientations accessible to UDG. These data indicate that the activity of UDG likely requires "trapping" transiently exposed states arising from the rotational dynamics of DNA on histones.

Fig. 1.

Uracil orientation in NCP substrates. Positioning of uracil residues in DNA on the surface of the histone octamer [figure created using PyMOL; structure taken from Davey et al. (54), Protein Data Bank Code 1KX5].

Fig. 2.

Hydroxyl radical footprint of reconstituted nucleosome core particles and relative uracil positions in the DNA substrate. Figure shows scans of a sequencing gel (Fig. S2) with the hydroxyl radical footprint of the undamaged DNA substrate associated with the histone octamer and the relative uracil positions of the U-In, U-Mid, and U-Out DNA constructs, after treatment with UDG and APE1. A labeled 10-bp DNA marker is also scanned for relative positioning, and the inverted triangle marks the dyad center of the NCP. Note that the actual position of the uracil residues of each substrate, in comparison with the NCP footprint, will be one nucleotide longer (shifted one peak to the left), as the APE1 endonuclease cleaves on the 5′ side of the lesion, between the uracil residue and the 5′ radiolabeled end.

Fig. 3.

Assessment of UDG and APE1 activity on NCP substrates with different uracil orientations. Plots of relative rates of cleavage of each DNA/NCP substrate (U-In, U-Mid, and U-Out in Upper, Middle, and Lower, respectively) over a 1-h time course. Dashed lines represent naked DNA of each construct; solid lines represent NCPs. UDG concentrations are listed on the Right Insets. For each construct, the data for naked DNA and NCPs incubated with 2 nM UDG is denoted by open boxes and open diamonds, respectively. Data for NCPs incubated with 20 nM UDG (For U-In and U-Mid only) are denoted by open triangles; data for naked DNA and NCPs incubated with 0.2 nM UDG (U-Out only) are denoted by open and closed circles, respectively. Each data point represents the mean ± 1 SD of at least three independent experiments.

Fig. 4.

UDG and APE1 cleavage of formaldehyde crosslinked NCPs. (Upper) Time course after Incubation with 0.2 nM UDG of Naked DNA (squares) and U-Out NCPs (triangles) with and without 1 h preincubation with formaldehyde (open symbols/dashed lines and closed symbols/solid lines, respectively). (Lower) Time course after incubation with 20 nM UDG of U-In (circles) and U-Mid NCPs (diamonds) with and without 1 h preincubation with formaldehyde (open symbols/dashed lines and closed symbols/solid lines, respectively). Each data point represents the mean ± 1 SD of at least three independent experiments.

Fig. 5.

Relative rates of hUDG and APE1-directed cleavage DNA and NCP constructs. Naked DNA (open triangles with dashed line) and NCP substrates U-In (small filled squares), U-mid (filled triangles), and U-Out (large filled squares) are represented. The hUDG concentration for the naked and U-Out NCPs is 2 nM, and for the U-In and U-Mid NCPs is 20 nM. Each data point represents the mean ± 1 SD of at least three independent experiments.

Fig. 6.

Influence of APE1 on hUDG activity in naked DNA and U-Out NCPs. Naked DNA (triangles and dashed lines) or U-Out NCPs (circles and solid lines) incubated with hUDG in the presence (filled symbols) or absence (open symbols) of APE1. Each data point represents the mean ± 1 SD of at least three independent experiments.