DNA polymerase family X: function, structure, and cellular roles

Abstract

The X family of DNA polymerases in eukaryotic cells consists of terminal transferase and DNA polymerases beta, lambda, and mu. These enzymes have similar structural portraits, yet different biochemical properties, especially in their interactions with DNA. None of these enzymes possesses a proofreading subdomain, and their intrinsic fidelity of DNA synthesis is much lower than that of a polymerase that functions in cellular DNA replication. In this review, we discuss the similarities and differences of three members of Family X: polymerases beta, lambda, and mu. We focus on biochemical mechanisms, structural variation, fidelity and lesion bypass mechanisms, and cellular roles. Remarkably, although these enzymes have similar three-dimensional structures, their biochemical properties and cellular functions differ in important ways that impact cellular function.

Figure 1. Alignment of Pol X Family Members

In red is the BRCT domain, purple is the 8 kDa domain, and green is the polymerase domain, consisting of thumb, palm, and fingers (not shown). The blue boxes represent Helix-Hairpin-Helix motifs, and the ones with the best conservation contain a star.

Figure 2. Repair Pathways

A. Base excision repair pathway. Light blue arrows connect steps in pathway started by a monofunctional DNA glycosylase. Dark blue arrows connect steps in pathway started by a binfunctional DNA glycosylase. Adapted from [48]. B. Non-homologous end joining pathway. Adapted from [177]. See text for detailed explanations.

Figure 3. Kinetic Pathway of DNA Polymerases β and λ

Schematic of catalytic scheme of pol β. Abbreviations: DNA_n, substrate DNA; DNA_n+1 extended product; Pol β* pol β in the transition state; PP_i pyrophosphate.

Figure 4. Crystal Structures of DNA Polymerase β

Crystal structures drawn using PDB 1BPY. 8kD domain is yellow, thumb subdomain is dark blue, palm subdomain is purple, fingers subdomain is teal, and the DNA is black. A. Overview of pol β. B. Close up view of the dNTP binding pocket. Residues of the dNTP binding pocket are shown as red sticks. The ddCTP is shown as green sticks. C. Close up view of other important dNTP binding residues. Key residues are shown as red sticks. ddCTP is shown as green sticks. D. Close up view of the hinge region of pol β. Hinge residues are shown as red sticks. ddCTP is shown as green sticks.

Figure 5. Pol β and 8oxoG

Structure of nucleotide insertions opposite an 8oxoG lesion. A. Chemical structure of an 8oxoG base in the anti configuration base paired with a dC. Adapted from [110]. B. Chemical structure of an 8oxoG base in the syn configuration base paired with a dA. Adaped from [110]. C. Crystal structure of pol β with an 8oxoG lesion in the active site opposite a dCTP from PDB 1MQ3. The 8oxoG and dCTP are colored by element. Pol β residue Lys280 is shown as red sticks. Subdomains are colored as for Figure 2.

Figure 6. Movement of Helix N upon Binding of a Mispair by Pol β

Cartoon representation of crystal structure of pol β with a mispair in the active site (PDB 3C2M). The mispaired crystal structure was aligned with crystal structures of the binary and correctly paired ternary complexes of pol β (PDB 1BPX and 1BPY, respectively). Only Helix N is shown of the 1BPX and 1BPY structures. The position of Helix N in the closed ternary complex with a correct pair is shown in blue, in the partially open ternary complex with a mispair is shown in purple, and in the open binary complex is shown in red.

Figure 7. Lesion Bypass by X Family Polymerases

The chemical structures of DNA lesions are shown along with X family polymerases capable of conducting bypass synthesis past them.