Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ

Abstract

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Evolution of Rev7 interactions in eukaryotic TLS DNA polymerase Polζ

“…This work is aimed to determine the consensus scRev3-RBMs and validate their interactions with scRev7, confirm the 2:1 stoichiometry of the scRev7:Rev3 complex, and investigate the scRev3-mediated scRev7 dimerization in solution. While we have validated the two adjacent RBMs within scRev3, our studies have not confirmed the novel head-to-tail dimerization of scRev7 observed in the recent cryo-EM structures of scPolζ ( 34 , 35 ), instead suggesting that scRev7 lacks the ability to form dimers in solution either alone or when tethered by scRev3-RBMs, which is a property that may have emerged later in evolution.…”

“…Although scRev7 binds scRev3 in a 2:1 stoichiometry, we found that scRev7 on its own is a monomer and have yet to obtain any data suggesting that scRev7 can homodimerize in solution when bound to scRev3-RBM12. Recent high-resolution cryo-EM structures corroborate a 2:1 scRev7:scRev3 stoichiometry within scPolζ with each scRev7 protomer binding to scRev3-RBM1 and scRev3-RBM2 regions ( 34 , 35 ). In contrast to hRev7, which forms symmetric head-to-head dimer through the canonical HORMA interface centered around helix αC ( 29 , 47 ), the cryo-EM structures (PDB: 6V8P , 6V93 , 7LXD ) suggest that scRev7 dimerizes asymmetrically in a head-to-tail fashion ( Fig.…”

“…In hPolζ, hRev3 was shown to bind two copies of human Rev7 (hRev7) via adjacent Rev7-binding motifs (RBMs), RBM1 and RBM2, with the consensus sequence Pxxx(A/P)P (where ‘x’ is any residue) ( 29 , 30 , 31 , 32 ), while the consensus sequence of RBMs within ∼250 amino acid scRev7-interacting region of scRev3 is yet to be established ( 10 , 20 , 27 , 33 ). Recent high-resolution cryo-EM structures of the scRev3:Rev7:Pol31:Pol32 assembly confirmed that scPolζ comprises two copies of scRev7 and narrowed the location of the two adjacent scRev3-RBMs ( 34 , 35 ) ( Fig. 1 ).…”

“…1 ). The structures reveal the scRev7:scRev3 interaction in a 2:1 stoichiometry, with the two scRev7 protomers forming an asymmetric head-to-tail dimer with a much smaller interface than the symmetric head-to-head hRev7 dimer or other dimers formed by HORMA protein pairs ( 34 , 35 ). The unexpected mode of scRev7 dimerization observed in the high-resolution cryo-EM structures of scPolζ ( 34 , 35 ) and conflicting earlier reports on scPolζ stoichiometry ( 20 , 21 ) highlight the potential differences in Polζ structural arrangements in higher and lower eukaryotes that warrant further investigation.…”

“…A breakthrough study of the scPolζ architecture reported a stoichiometric complex of scRev3:scRev7:Pol31:Pol32 subunits at 1:1:1:1 ratio ( 21 ), and the initial low-resolution electron microscopy (EM) structure of the four-subunit complex was modeled with one copy of scRev7 ( 20 ), posing the question whether the Rev7 dimerization is a distinct property of the Polζ assembly in higher eukaryotes. Recently, three high-resolution cryo-EM structures of scPolζ were reported (PDB: 6V8P , 6V93 , 7LXD ), a remarkable feat that describes the spatial organization of scPolζ subunits and a variety of protein interactions within the scPolζ complex ( 34 , 35 ) ( Fig. 1 ).…”