Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ

The translesion synthesis (TLS) DNA polymerases Rev1 and Pol zeta function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Pol zeta in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA-and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Pol zeta holoenzyme. We show that compared with its DNA-bound state, apo Pol zeta displays enhanced flexibility that correlates with concerted motions associated with expansion of the Pol zeta DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Pol zeta, suggesting a gating mechanism. The Pol zeta subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Pol zeta and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Pol zeta mechanism of action and the manner in which Rev7 recognizes partner proteins.

Automated summary

The study revealed that apo Pol zeta exhibits enhanced flexibility compared to its DNA-bound state, with concerted motions associated with expansion of the Pol zeta DNA-binding channel upon DNA binding. Additionally, a lysine residue obstructing the DNA-binding channel in apo Pol zeta was identified, suggesting a gating mechanism.