Eukaryotic replicative DNA polymerases (Pols), Pols δ and ε belong to the B-family of Pols and they replicate DNA with a very high fidelity. Although DNA polymerase ζ (Polζ) is also a member of the B-family, it differs from the replicative Pols in that it synthesizes DNA with a lower fidelity, and plays a critical role in promoting replication through a wide variety of DNA lesions. Polζ is unique in this regard. Catalytically active Polζ is comprised of the Rev3 catalytic and Rev7 accessory subunits; however, we showed that the polymerase contains two additional subunits in vivo, Pol31 and Pol32, and we refer to this four-subunit complex as Polζ-d. Moreover, we have shown recently that Rev1, a member of the Y-family of Pols, is also a stoichiometric component of Polζ-d, and we refer to this five-subunit complex as Polζ-d1. Our ability to purify Polζ-d and Polζ- d1 opens up these multi-subunit complexes for a detailed mechanistic and structural analysis. We are able to ask for the first time questions related to the overall architecture of Polζ-d and Polζ−d1 and how Pol31, Pol32, and Rev1 potentiate the catalytic activity of Polζ on undamaged and damaged DNA substrates. In Aim 1, we will carry out cryo-electron microscopy (cryo-EM) analysis of Polζ-d and Polζ-d1 in the presence of DNA, taking advantage of the latest developments in direct detection device cameras for imaging single molecules and software for 3-D reconstruction at high-resolution. These studies will build on our earlier low-resolution model of Polζ-d and reveal for the first time how the enzyme actually interacts with DNA and the nature of protein- protein contacts between the various subunits. In Aim 2, we will carry out pre-steady-state kinetic analyses to determine the action mechanisms of Polζ-d, and Polζ-d1. These studies will be performed in conjunction with the structural studies (Aim 1) to obtain a kinetic picture of the reaction pathway. We will also test the structures by biochemical and genetic approaches, whereby residues in the Rev3 active site and those that mediate subunit protein-protein interactions will be mutated and assayed for their effect on DNA synthesis in vitro and on DNA damage response in vivo. Altogether, the proposed studies are important for understanding how eukaryotic cells cope with a diverse array of DNA lesions induced by exogenous and endogenous genotoxic agents.