The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

Abstract

In their seminal publication describing the structure of the DNA double helix, Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.

Figure 1. Determinants of replication fidelity

A. The relative contribution levels of the three main components of replication fidelity are shown above the scale, estimated from the mutation rates of systems defective in one or more of the components. The overlapping ovals represent the fact that there is a range of possible increases in the level of fidelity that each mechanism provides dependent on many factors. The range of fidelity that a given mechanism is capable of providing is the critical factor (i.e. MMR can still provide up to 4 orders of magnitude increase in fidelity for polymerase errors that occur at a frequency of 10⁻²). The horizontal bars below the graph show the ranges of in vitro determined error rates for the different families of polymerases and the estimated mutation rate range of the in vivo complete replication complex. Within each family, the error rates can differ widely between polymerases and type of error. The broken bars at the left and right ends indicate that the rates could be even higher and lower than indicated. B. Graphic depicting the various means by which DNA replication can be modulated. DNA is shown as a stylized double helix (backbone is black and gray), with purine-pyrimidine base pairs indicated as red-green and blue-purple bars. The single strand region is meant to depict the unwound DNA at a replication fork, with the kink in the DNA representing the bend in the template strand identified by crystallography. Red arrows and text indicate conditions that lead to lower fidelity. Green arrows and text indicate conditions that promote higher fidelity, green bars indicate conditions that block mutations. M = Mutation; C = Correct.

Figure 2. Simplified model of replication fork

A cartoon model of eukaryotic replication fork. Protein depictions are based on currently accepted sub-unit composition of S. cerevisiae proteins but are not meant to be accurate structure-based models. The assignment of pol ε to the leading strand is based on a recent report, but has not been definitively established for all replication. Pol δ is consequently assigned to the lagging strand, consistent with earlier reports^–. Helicase hexamer (magenta); Replication protein A (RPA; light blue ovals); Proliferating Cell Nuclear Antigen (PCNA; purple torus); pol α-primase complex (blue); RNA-DNA hybrid primer (red zig-zag and arrow); pol δ (red); pol ε (green); Template strand DNA (black lines); Newly synthesized DNA (gray lines). Figure inspired by and adapted from Figure 1 in reference and Figure 7 reference.

Figure 3. Models of translesion synthesis

A. The 1-polymerase model of TLS, shown here for a thymine-thymine dimer, states that a single polymerase is responsible for the complete bypass of a lesion, including insertion opposite all lesion bases and extension from the primer termini opposite a damaged template base. B. The 2-polymerase model of TLS, shown here for a thymine-thymine 6-4 photoproduct, states that different polymerases are responsible for the insertion steps at the various lesion positions. In the example given, note that while pol ζ is responsible for extension from the template-3´T primer terminus, it is also an insertion at the 5´ T position of the lesion. For a single base lesion, the insertion step would be opposite undamaged DNA. A more comprehensive listing of 2-polymerase/lesion combinations is given elsewhere. Note that for both examples given the actual TLS reaction is flanked relatively closely both upstream (1–2 bases) and downstream (1–5 bases) of the lesion by replicative polymerase synthesis. C. Model for TLS that occurs at a replication fork during the process of ongoing synthesis. D. Model for TLS that takes place as a ‘gap-filling’ reaction, away from the main replication machinery. Note that both of these models are consistent with either the 1- or 2-polymerase model of TLS given in Panels A and B. In both cases, post-translational modification of PCNA and possible other proteins is critical for the polymerase switch. Note that Panels A and B are models of the actual TLS process while Panels C and D depict models for the timing of TLS. As such (and as noted in the text), there is overlap between the panels.

Figure 4. Open and closed active sites of low and high fidelity polymerases

A and B. Shown are the molecular surfaces of Dpo4 from S. solfataricus and T7 DNA polymerase with blue representing positively charged regions and red representing negatively charged regions. Note the tighter fit of DNA (ball and stick model) to the higher fidelity T7 DNA polymerase, evidenced by closer contact with polymerase regions. In Dpo4, the DNA is located at further distance from the polymerase, and a ‘hole’ in the polymerases structure is visible, indicating a much looser association of the DNA with the polymerase. C and D. Closeup of the active site showing the templating base and nucleotide. Again, note the relatively open and solvent accessible region in Dpo4 compared to the snug fit in T7 DNA polymerase. Also note the increased amount of neutral region of protein in T7 DNA polymerase, indicating that it is the geometry of the replicative polymerase active site that plays a role in fidelity, as noted in the text. This figure appears originally as Figure 7 in and has been reproduced with the permission of the authors.