Abstract
Translesion synthesis (TLS) is the mechanism by which DNA polymerases replicate through unrepaired DNA lesions. TLS is activated by monoubiquitination of the homotrimeric proliferating cell nuclear antigen (PCNA) at lysine-164, followed by the switch from replicative to specialized polymerases at DNA damage sites. Pol η belongs to the Y-Family of specialized polymerases that can efficiently bypass UV-induced lesions. Like other members of the Y-Family polymerases, its recruitment to the damaged sites is mediated by the interaction with monoubiquitinated PCNA (Ub-PCNA) via its ubiquitin-binding domain and non-canonical PCNA-interacting motif in the C-terminal region. The structural determinants underlying the direct recognition of Ub-PCNA by Pol η, or Y-Family polymerases in general, remain largely unknown. Here we report a structure of the Ub-PCNA/Pol η complex bound to DNA determined by single-particle electron microscopy (EM). The overall obtained structure resembles that of the editing PCNA/PolB complex. Analysis of the map revealed the conformation of ubiquitin that binds the C-terminal domain of Pol η. Our present study suggests that the Ub-PCNA/Pol η interaction requires the formation of a structured binding interface, which is dictated by the inherent flexibility of Ub-PCNA.
Highlights
The integrity of the genome is constantly challenged by endogenous and exogenous DNA-damage agents[1,2]
Following the purification of Ub-proliferating cell nuclear antigen (PCNA) and Pol η, a binding assay was carried out to confirm their assembly onto a DNA template-primer substrate (Supplementary Fig. S1)
Monoubiquitination of PCNA represents the key signal for the switch between high-fidelity and specialized DNA polymerases to bypass a variety of DNA lesions at stalled replication sites
Summary
The integrity of the genome is constantly challenged by endogenous and exogenous DNA-damage agents[1,2]. The Rad6-Rad[18] ubiquitin conjugating/ligase complex mediates the monoubiquitination of PCNA, which in turn recruits TLS polymerases to the sites of DNA lesions[19,21]. The C-terminal, non-catalytic domain of Pol η contains multiple motifs and regions including the PIP-box and the UBD (called UBZ domain in Pol η ) as mentioned above for interactions with partner proteins[7,26]. This region was predicted to be mostly unstructured based on bioinformatics analysis but may undergo disordered-to-ordered transition upon binding to its cognate partner proteins[30]. Our structural data unveil the distinct ubiquitin conformation recognized by Pol η and indicate that intrinsic flexibility of the ubiquitin on PCNA provides the accessible surface for its binding partners
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