Abstract
DNA synthesis across lesions during genomic replication requires concerted actions of specialized DNA polymerases in a potentially mutagenic process known as translesion synthesis. Current models suggest that translesion synthesis in mammalian cells is achieved in two sequential steps, with a Y-family DNA polymerase (κ, η, ι, or Rev1) inserting a nucleotide opposite the lesion and with the heterodimeric B-family polymerase ζ, consisting of the catalytic Rev3 subunit and the accessory Rev7 subunit, replacing the insertion polymerase to carry out primer extension past the lesion. Effective translesion synthesis in vertebrates requires the scaffolding function of the C-terminal domain (CTD) of Rev1 that interacts with the Rev1-interacting region of polymerases κ, η, and ι and with the Rev7 subunit of polymerase ζ. We report the purification and structure determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol ζ complex, and the Pol κ Rev1-interacting region. Yeast two-hybrid assays were employed to identify important interface residues of the translesion polymerase complex. The structural elucidation of such a quaternary translesion polymerase complex encompassing both insertion and extension polymerases bridged by the Rev1 CTD provides the first molecular explanation of the essential scaffolding function of Rev1 and highlights the Rev1 CTD as a promising target for developing novel cancer therapeutics to suppress translesion synthesis. Our studies support the notion that vertebrate insertion and extension polymerases could structurally cooperate within a megatranslesion polymerase complex (translesionsome) nucleated by Rev1 to achieve efficient lesion bypass without incurring an additional switching mechanism.
Highlights
Translesion synthesis in mammalian cells is achieved by sequential actions of insertion and extension polymerases
To more fully characterize the Rev1-Rev[7] interaction, we explored the requirement of specific amino acids of Rev[7] that reside at the Rev1-Rev[7] interface in promoting an interaction between these two proteins
We confirmed that the K1199E mutant Rev[1] protein is expressed at a level similar to that of wild-type Rev[1], demonstrating that the K1199E mutation does not drastically destabilize the protein (Fig. 6B). These results suggest that the specific interaction between the Rev[1] C-terminal domain (CTD) and Rev[7], characterized using yeast two-hybrid assays in our previous study (20) and in this paper, is critical for the function of the Rev1-Pol complex in vertebrate cells that have suffered DNA damage
Summary
Translesion synthesis in mammalian cells is achieved by sequential actions of insertion and extension polymerases. Despite the presence of highly efficient DNA repair processes in cells, a small number of lesions inevitably evade the surveillance of sophisticated repair machinery and block the progression of high fidelity replicases during genomic replication, resulting in arrested replication forks and generation of single-stranded replication gaps. Both of these events contribute to genome instability and pose a serious challenge for cell viability. REV1, REV3, and REV7 were the first translesion polymerase genes identified based on mutations that resulted in significantly reduced muta-
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