Evolving views of DNA replication (in)fidelity

Abstract

"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material" (Watson and Crick 1953). In the years since this remarkable understatement, we have come to realize the enormous complexity of the cellular machinery devoted to replicating DNA with the accuracy needed to maintain genetic information over many generations, balanced by the emergence of mutations on which selection can act. This complexity is partly based on the need to remove or tolerate cytotoxic and mutagenic lesions in DNA generated by environmental stress. Considered here is the fidelity with which undamaged and damaged DNA is replicated by the many DNA polymerases now known to exist. Some of these seriously violate Watson-Crick base-pairing rules such that, depending on the polymerase, the composition and location of the error, and the ability to correct errors (or not), DNA synthesis error rates can vary by more than a millionfold. This offers the potential to modulate rates of point mutations over a wide range, with consequences that can be either deleterious or beneficial.

Figure 1. X ray crystal structures of DNA polymerases

A. Shown is the structure of a representative replicative DNA polymerase from bacteriophage RB69 (family B). Polymerase domains share three common sub-domains, designated fingers (blue), palm (red) and thumb (green). Other domains for specialized functions are shown in purple and yellow. (B) The active site of human DNA polymerase β. The surface of Arg283 is highlighted in pink to emphasize the importance to fidelity of polymerase interactions with the DNA minor groove. (C) The more open and solvent-accessible active site of low-fidelity Sulfolobus sulfataricus Dpo4. See text for further descriptions. Panel (A) was prepared by Miguel Garcia-Diaz, using the structure in (Franklin et al., 2001). Panel (B) and (C) are reproduced from (Kunkel et al., 2003), with permission.

Figure 2. Polymerase error rates and the contributions of each fidelity process to mutation rate

The image illustrates the wide ranges over which polymerase nucleotide selectivity, exonucleolytic proofreading and mismatch repair contribute to spontaneous mutation rates of organisms. Also depicted are the average rates at which purified eukaryotic DNA polymerases generate single base substitution and single base deletion errors when performing gap-filing DNA synthesis in vitro. See text for further descriptions. Details on the source and composition of the polymerases used, and on their error specificity, can be found in (McCulloch and Kunkel, 2008) and references therein.

Figure 3. Exonucleolytic proofreading

(A) Depiction of the principles that determine the efficiency of proofreading. A proofreading-proficient polymerase (blue) harbors its polymerase and exonuclease activities in separate domains (e.g., see RB69 pol in Fig. 1A), depicted as large and small ovals, respectively. The partitioning between these two activities determines the efficiency of proofreading. Also shown is the possibility that errors made by an exonuclease-deficient polymerase (yellow) may be proofread by a separate exonuclease, either that of a proofreading-proficient polymerase (as shown) of present in another protein. See text for a further description and (Nick McElhinny et al., 2006) and references therein] for additional discussion and information. (B) The left panel depicts the single base deletion error rates of proofreading-proficient (open bars) and proofreading-deficient (closed bars) T7 DNA polymerase when copying tracks of 3–8 consecutive template Ts. The right panel depicts the ratio of the error rates of the two polymerases, to illustrate the decreasing efficiency of proofreading as a function of increasing repetitive sequence track length. Reproduced from (Kroutil and Kunkel, 1998), with permission.

Figure 4. Replication fork and translesion synthesis models

(A) Current model of the eukaryotic DNA replication fork (left), with pol ε replicating the leading strand template and pols α and δ replicating the lagging strand template. On the right is an “alternative fork” that might result from stress. See text and (Kunkel and Burgers, 2008) for further discussion. Reproduced from (Kunkel and Burgers, 2008), with permission. (B) “One TLS polymerase” (left) and “Two TLS polymerase” (right) models for translesion DNA synthesis. See text for further discussion.

Figure 5. DNA mismatch repair

Models for DNA mismatch repair in E. coli (A) and eukaryotes (B). See text and for description. Reproduced from (Iyer et al., 2006), with permission.