Variations on a theme: eukaryotic Y-family DNA polymerases

Abstract

Most classical DNA polymerases, which function in normal DNA replication and repair, are unable to synthesize DNA opposite damage in the template strand. Thus in order to replicate through sites of DNA damage, cells are equipped with a variety of nonclassical DNA polymerases. These nonclassical polymerases differ from their classical counterparts in at least two important respects. First, nonclassical polymerases are able to efficiently incorporate nucleotides opposite DNA lesions while classical polymerases are generally not. Second, nonclassical polymerases synthesize DNA with a substantially lower fidelity than do classical polymerases. Many nonclassical polymerases are members of the Y-family of DNA polymerases, and this article focuses on the mechanisms of the four eukaryotic members of this family: polymerase eta, polymerase kappa, polymerase iota, and the Rev1 protein. We discuss the mechanisms of these enzymes at the kinetic and structural levels with a particular emphasis on how they accommodate damaged DNA substrates. Work over the last decade has shown that the mechanisms of these nonclassical polymerases are fascinating variations of the mechanism of the classical polymerases. The mechanisms of polymerases eta and kappa represent rather minor variations, while the mechanisms of polymerase iota and the Rev1 protein represent rather major variations. These minor and major variations all accomplish the same goal: they allow the nonclassical polymerases to circumvent the problems posed by the template DNA lesion.

Fig. 1

Mechanism of DNA synthesis by DNA polymerases. The minimal mechanism of nucleotide incorporation by all DNA polymerases contains at least six steps. Step 1: the polymerase binds the DNA substrate. Step 2: the polymerase-DNA binary complex binds the incoming dNTP substrate. Step 3: the bound nucleotide is incorporated. Step 4: the pyrophosphate (PP_i) product is released. Step 5: the polymerases translocates along the DNA. Step 6: alternatively, the polymerase dissociates from the DNA.

Fig. 2

The polymerase switching mechanism. Step 1: the classical polymerase (purple) dissociates from the stalled replication complex. Step 2: the non-classical polymerase (blue) associates with the stalled complex. Step 3: the non-classical polymerase catalyzes nucleotide incorporation opposite the lesion and further extension. Step 4: the non-classical polymerase dissociates from the replication complex. Step 5: the classical polymerase returns to the replication complex.

Fig. 3

The active site of pol η. The structure of the pol η (light blue) determined in the absence of DNA and dNTP substrates with DNA (orange) containing a template thymine dimer (yellow) and an incoming dATP (green) modeled into the active site is shown. This model is based on a previously published model [51]. The oxygen (red) and nitrogen (blue) atoms on the incoming nucleotide and template residue are shown. The Watson-Crick hydrogen bonds are indicated by dashed lines. (PDB ID: 1JIH, reference [51])

Fig. 4

The active site of pol κ. Pol κ (light blue) is shown bound to DNA (orange) with A (yellow) as the template and dTTP (green) as the incoming nucleotide. The oxygen (red) and nitrogen (blue) atoms on the incoming nucleotide and template residue are shown. The Watson-Crick hydrogen bonds are indicated by dashed lines (PDB ID: 2OH2, reference [80])

Fig. 5

The active site of pol ι. Pol ι (light blue) is shown bound to DNA (orange) with 1,N⁶-etheno-A template (yellow) as the template and dTTP (green) as the incoming nucleotide. The anti or syn conformations of the glycosidic bond are indicated. The oxygen (red) and nitrogen (blue) atoms on the incoming nucleotide and template residue are shown. The Hoogsteen hydrogen bonds are indicated by dashed lines (PDB ID: 2DPJ, reference [85])

Fig. 6

The active site of the Rev1 protein. The Rev1 protein (light blue) is shown bound to DNA (orange) with 1,N²-propano-G (yellow) as the template and dCTP (green) as the incoming nucleotide. The side chain of Arg-324 (purple) is shown. The oxygen (red) and nitrogen (blue) atoms on the incoming nucleotide, template residue, and Arg-324 are shown. The hydrogen bonds between the incoming dCTP, the DNA, and the side chain of Arg-324 are indicated by dashed lines (PDB ID: 3BJY, reference [119])