Eukaryotic translesion synthesis: Choosing the right tool for the job

Abstract

Normal DNA replication is blocked by DNA damage in the template strand. Translesion synthesis is a major pathway for overcoming these replication blocks. In this process, multiple non-classical DNA polymerases are thought to form a complex at the stalled replication fork that we refer to as the mutasome. This hypothetical multi-protein complex is structurally organized by the replication accessory factor PCNA and the non-classical polymerase Rev1. One of the non-classical polymerases within this complex then catalyzes replication through the damage. Each non-classical polymerase has one or more cognate lesions, which the enzyme bypasses with high accuracy and efficiency. Thus, the accuracy and efficiency of translesion synthesis depends on which non-classical polymerase is chosen to bypass the damage. In this review article, we discuss how the most appropriate polymerase is chosen. In so doing, we examine the structural motifs that mediate the protein interactions in the mutasome; the multiple architectures that the mutasome can adopt, such as PCNA tool belts and Rev1 bridges; the intrinsically disordered regions that tether the polymerases to PCNA and to one another; and the kinetic selection model in which the most appropriate polymerase is chosen via a competition among the multiple polymerases within the mutasome.

Keywords: DNA polymerase; DNA repair; DNA replication; Genome instability; Mutasome; PCNA; Protein-protein interactions; Structure.

Figure 1.. Assembly of the mutasome.

The model of PCNA (blue) was obtained from the X-ray crystal structure (1PLQ). The model of full-length Rev1 (green) was obtained from X-ray crystal structures of the BRCT domain (4ID3), the polymerase domain (3OSP), and the CTD (3VU7 and 2LSK). The model of pol ζ (red) was obtained from the X-ray crystal structure of pol δ (3IAY). The model of pol η (orange) was obtained from the X-ray crystal structure the polymerase domain (5VTP). Pol ζ and pol η bind PCNA via their PIP motifs (insets A and B). Rev1 binds PCNA via the Rev1 BRCT domain (inset C). Pol ζ binds the Rev1 CTD via the Rev7 subunit (inset D). Pol η binds the Rev1 CTD via its PIP motif (inset E).

Figure 2.. Tethering of pol η and Rev1 to PCNA.

An overlay of ten structures of full-length pol η (orange) from the ensemble obtained from BD simulations (left). These the PIP motifs of these structures have been aligned and placed in the PIP motif-binding site of PCNA (blue). An overlay of ten structures of full-length Rev1 (green) from the ensemble obtained from BD simulations (right). The BRCT domains of these structures have been aligned and placed in the BRCT domain-binding site of PCNA (blue).

Figure 3.. The kinetic selection model.

A complex containing PCNA (blue), Rev1 (green), pol η (orange), and DNA (white) organized as a PCNA tool belt is shown. Initially, neither non-classical polymerase in this complex is engaged with the DNA substrate. Either pol η engages the DNA substrate (upper pathway) or Rev1 engages the DNA substrate (lower pathway). Once the polymerases have engaged the DNA substrate, they may incorporate one or more nucleotides. The kinetic constants are defined in the text.

Figure 4.. BD simulations of PCNA tool belts and Rev1 polymerase bridges.

We carried out 96 BD simulations of PCNA tool belts (left) and 96 BD simulations of Rev1 polymerase bridges (right). These complexes contained PCNA (blue), Rev1 (green), pol η (orange), and DNA (white). In these simulations, each amino acid residue or nucleotide was represented by one CG bead. The simulations were run with a time step of 125 fs, and a pdb file was generated every 1 ns. Each simulation was run for a total of 500 ns. The engagement of a polymerase was defined to be a contact between the primer-terminus of the DNA and any surface of the polymerase that persisted until the end of the simulation. The percentages of simulations in which Rev1 engaged the DNA substrate and in which pol η engaged the DNA substrate are indicated.