Structural basis for the suppression of skin cancers by DNA polymerase eta

Abstract

DNA polymerase eta (Poleta) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of Poleta (also known as POLH) in humans causes the variant form of xeroderma pigmentosum (XPV). We present the crystal structures of Saccharomyces cerevisiae Poleta (also known as RAD30) in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures reveal that the ability of Poleta to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the two Ts of the T-T dimer are accommodated in an active site cleft that is much more open than in other polymerases. We also show by structural, biochemical and genetic analysis that the two Ts are maintained in a stable configuration in the active site via interactions with Gln 55, Arg 73 and Met 74. Together, these features define the basis for Poleta's action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans.

Figure 1

Polη-DNA-dATP ternary complexes. a, Structure of Polη with undamaged DNA. The palm, fingers, thumb domains and the PAD are shown in cyan, yellow, orange and green, respectively. The DNA is in gray, and the putative Mg²⁺ ions are in dark blue. The templating 3'T (and the 5'T) and incoming dATP are in red. b, Structure of Polη with a cis-syn T-T dimer in DNA. The cis-syn T-T dimer and incoming dATP are shown in red. Yellow and orange dashed lines depict unstructured loops in the fingers and thumb domains, respectively.

Figure 2

Close-up views of the active site regions. a, The active site regions with undamaged (left) and T-T dimer-containing (right) DNAs. The palm and fingers domains and the PAD are shown in cyan, yellow, and green, respectively. The DNA is colored gray, and the putative Mg²⁺ ions (A and B) are dark blue. The undamaged templating T (and the 5'T) (left), the cis-syn T-T dimer (3'T and 5'T), and incoming dATP are in red. Highlighted and labeled are the catalytic residues (D30, D155, and E156), residues that interact with the triphosphate moiety of incoming dATP (Y64, R67, and K279), residues that interact with templating T and the 5'T (Q55, W56, M74, and R73), and F35, which stacks against the dATP sugar. R73 is shown in two orientations in the T-T dimer structure. Y452 is also shown (right), which stacks against the base of the nucleotide 5' to the T-T dimer. The residues are colored to match the domain they belong to. b, Simulated annealing Fo-Fc omit maps (contoured at 3.0σ) of undamaged templating T (and the 5'T) and incoming dATP (left), and cis-syn T-T dimer and incoming dATP (right). c, Hydrogen bonding and van der Waals interactions between residues on the fingers domain (Q55, W56, I60, R73 and M74) and undamaged Ts (left) and cis-syn T-T dimer (right).

Figure 3

Conformational changes in Polη ternary complex. a, Conformational change in Polη upon complex formation. Upon DNA and dNTP binding, the PAD swings by as much as ~13.6A towards the major groove of the DNA. The thumb also rotates towards the DNA but to a much lesser extent than the PAD. b, Comparison between our Polη ternary complex and that reported by Alt et al between Polη and a cisplatin adduct. In the Alt et al. structure, Polη has an apo-like conformation (cf Figure 3a), wherein the PAD occupies a position similar to that in the apo Polη structure

Figure 4

Comparison between Polη and Polκ. a, Polκ (gray) superimposed on the Polη ternary complex via the palm domain. In Polκ, the fingers domain is in close proximity to the cis-syn T-T dimer (red) and it collides with the 5'T of the T-T dimer. b, Left, molecular surface of Polη in the UV-damaged ternary complex. Both Ts of the T-T dimer fit unhindered within the active site cleft. Right, molecular surface of Polκ when superimposed on the Polη ternary complex via the palm domain. The 5'T of the T-T dimer collides with M135 and other residues of the Polκ fingers domain.