Human 8-oxoguanine glycosylase OGG1 binds nucleosome at the dsDNA ends and the super-helical locations

Abstract

The human glycosylase OGG1 extrudes and excises the oxidized DNA base 8-oxoguanine (8-oxoG) to initiate base excision repair and plays important roles in many pathological conditions such as cancer, inflammation, and neurodegenerative diseases. Previous structural studies have used a truncated protein and short linear DNA, so it has been unclear how full-length OGG1 operates on longer DNA or on nucleosomes. Here we report cryo-EM structures of human OGG1 bound to a 35-bp long DNA containing an 8-oxoG within an unmethylated Cp-8-oxoG dinucleotide as well as to a nucleosome with an 8-oxoG at super-helical location (SHL)-5. The 8-oxoG in the linear DNA is flipped out by OGG1, consistent with previous crystallographic findings with a 15-bp DNA. OGG1 preferentially binds near dsDNA ends at the nucleosomal entry/exit sites. Such preference may underlie the enzyme's function in DNA double-strand break repair. Unexpectedly, we find that OGG1 bends the nucleosomal entry DNA, flips an undamaged guanine, and binds to internal nucleosomal DNA sites such as SHL-5 and SHL+6. We suggest that the DNA base search mechanism by OGG1 may be chromatin context-dependent and that OGG1 may partner with chromatin remodelers to excise 8-oxoG at the nucleosomal internal sites.

Fig. 1. Cryo-EM structure of the OGG1 bound to a long naked DNA.

a Domain architecture of the human OGG1. b Representative 2D class averages. c Cryo-EM structure shown in two side views, with the EM map superimposed and shown as transparent surface in the left panel. d The catalytic site of cryo-EM structure using a 35-bp DNA duplex reveals a flipped 8-oxoG. The interaction between the OGG1 N151 and the DNA C (+17) base, and the flipped 8-oxoG base are highlighted. e A schematic drawing of OGG1–DNA interaction. f Comparison of DNA bending in the OGG1–DNA EM structure and previous crystal structure. Inserted below is a comparison of the top sequence (TS) and bottom sequence (BS) of the DNA used in the current EM study and the previous crystal structure (PDB ID 1EBM).

Fig. 2. OGG1 C-terminal extension interacts with DNA.

a The EM density of the OGG1 C-tail helix (magenta) adjacent to the DNA backbone. The 3D EM map is surface rendered at a very low threshold to visualize the weak C-tail helix density. The low threshold rendering makes the EM map appear at a lower resolution than the actual resolution of 3.6 Å. b The AphaFold2 prediction for the C-terminal of OGG1 shows a longer α-helix than the previous crystal structure assigned, which is consistent with the C-terminal extension density in OGG1–DNA cryo-EM 3D map. c Predicted additional DNA interaction region in OGG1.

Fig. 3. More than one OGG1 can bind to the single 8-oxoG-containing DNA, and the binding ability is reduced in the C-terminus truncated OGG1.

a Formation of the OGG1-DNA complex was monitored by EMSA. 2.5 μM of 35-mer DNA duplex containing 8-oxoG at the middle base was mixed with OGG1 at the indicated concentrations. b EMSA was carried out with 8-oxoG DNA duplex using C-terminus deleted OGG1 (OGG1-$△$CT) at the indicated concentrations. c SDS-PAGE gel of purified OGG1 and OGG1-$△$CT used in the EMSA assay. d Four selected 2D class averages of two OGG1 molecules bound to the same linear DNA. The 35-bp dsDNA is bent twice, at the middle 8-oxoG (red dot) and near the end with a normal DNA base (white dot).

Fig. 4. Structure of the nucleosome containing an 8-oxoG at the SHL-5 site.

a Cryo-EM 3D map. b Superposition of the nucleosome with 8-oxoG and a canonical nucleosome (cyan, PDB ID 6FQ5). The right panel is a close-up view of the boxed 8-oxoG region around the SHL-5, superimposed with the EM density in transparent gray surface, showing that DNA structure at the 8-oxoG is not distorted and is virtually identical to the canonical nucleosomal DNA.

Fig. 5. Cryo-EM structures of the OGG1-nucleosome (8-oxoG) in two distinct complexes.

a, b Cryo-EM 3D maps of the OGG1–nucleosome in complex I (a) and complex II (b). The right panels show representative 2D averages of these two complexes. c, d Rigid-body fitting of the nucleosome (8-oxoG) structure into the 3D maps of complex I (c) and complex II (d). The right panels show the respective close-up views around the SHL DNA density where OGG1 binds. The nucleosomal DNA appears unaltered by OGG1 binding at either SHL-5 or SHL+6 sites.

Fig. 6. OGG1 binding at the nucleosome entry site bends DNA and flips a guanosine base.

a Selected 2D class averages of nucleosome particles with clear OGG1 binding at the entry site. b Composite EM map of the OGG1–nucleosome complex. The histone core and DNA are resolved to 3.2 Å, but the OGG1 is at a lower resolution of 5–7 Å. c Atomic model of the nucleosome with OGG1 at the entry site in cartoons. The zoomed windows show that OGG1 bends the local DNA and flips the G near the end of the nucleosome DNA. d A proposed OGG1 interrogation mechanism. OGG1 slides rapidly in unobstructed linear DNA regions. In the fast-sliding mode, OGG1 searches for 8-oxoG without flipping the bases. In highly crowded regions where DNA is packed into nucleosomes and bound by additional chromatin proteins, OGG1 may move slowly. And in the slow sliding mode, OGG1 flips the bases to search for 8-oxoG.