3,4 ✉ mHsp60-mHsp10 assists the folding of mitochondrial matrix proteins without the negative ATP binding inter-ring cooperativity of GroEL-GroES. Here we report the crystal structure of an ATP (ADP:BeF 3 -bound) ground-state mimic double-ring mHsp60 14 -(mHsp10 7 ) 2 football complex, and the cryo-EM structures of the ADP-bound successor mHsp60 14 -(mHsp10 7 ) 2 complex, and a single-ring mHsp60 7 -mHsp10 7 half-football. The structures explain the nucleotide dependence of mHsp60 ring formation, and reveal an inter-ring nucleotide symmetry consistent with the absence of negative cooperativity. In the ground-state a two-fold symmetric H-bond and a salt bridge stitch the double-rings together, whereas only the H-bond remains as the equatorial gap increases in an ADP football poised to split into halffootballs. Refolding assays demonstrate obligate single-and double-ring mHsp60 variants are active, and complementation analysis in bacteria shows the single-ring variant is as efficient as wild-type mHsp60. Our work provides a structural basis for active single-and double-ring complexes coexisting in the mHsp60-mHsp10 chaperonin reaction cycle.
DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, and yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of
S.cerevisiae
Polζ holoenzyme in the act of DNA synthesis (3.1Å) and without DNA (4.1Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3 and accessory Pol31, Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein-protein interactions. We also uncover the features that impose high fidelity during the nucleotide incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ’s role in TLS and provide a framework for new cancer therapeutics.
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