Pools and Pols: Mechanism of a mutator phenotype.

The maintenance of the human genome is dependent upon several cellular processes including DNA replication. Ordinarily, DNA replication is an exceptionally faithful process, with approximately one error occurring for every 10–10 nucleotides (1, 2). High-fidelity replicative DNA polymerases with exonucleolytic proofreading activity, along with DNA mismatch repair machinery, are responsible for accurate DNA synthesis. DNA polymerase δ (pol δ) and polymerase e (pol e) are two essential replicative lagging and leading strand polymerases, respectively, that ensure efficient and high-fidelity genome replication (3–5). Replicative polymerase variants have recently been identified in human tumors that harbor enormous numbers of mutations. In recent studies reported in PNAS, Mertz et al. and Williams et al. provide evidence that this hypermutator phenotype results from expansion of deoxyribonucleoside triphosphate (dNTP) pools (6, 7). The concept of a mutator phenotype leading to cancer was introduced by Lawrence Loeb in 1974 to account for the disparity between the low mutation rates in normal cells and the large numbers of mutations present in many human tumors. This hypothesis has evolved over time, with better understanding of the molecular basis for tumorigenesis, and it is now known that a mutator phenotype results from mutations in genes that maintain genome stability (8–13). The recently identified germ-line and somatic mutations of POLE and POLD, which encode DNA polymerases e and δ, respectively, and which are associated with hypermutated tumors, provide additional strong evidence for the mutator phenotype hypothesis. Mutator variants of pol e and pol δ have recently been characterized in Saccharomyces cerevisiae by Williams et al. and Mertz et al. (6, 7). Williams et al. characterized the pol2-4 proofreading defective and pol2-M644G base selectivity-deficient alleles of pol2, which encode pol e (5, 14). Mertz and colleagues studied the pol3-R696W S. cerevisiae mimic of a POLD variant that is deficient in base selectivity that was identified in two colorectal cancer cell lines, DLD1 and HCT15, which were derived from the same tumor (15–17). Interestingly, Williams et al. and Mertz et al. find the mutator phenotypes of these POLE and POLD variants depend upon the Dun1 effector kinase, which plays a role in the regulation of dNTP pools and the transcriptional response to DNA damage (Fig. 1) (6, 7, 18). In fact, cells with the pol2-M644G allele are dependent upon Dun1 for survival. It has been well established that dNTP pools are precisely regulated during the cell cycle by ribonucleotide reductase (RNR) (19, 20). In addition, Chabes et al. demonstrated that dNTP levels rise sixto eightfold as a result ofDNAdamage (21). These increased pools were a consequenceof activationof aDNAdamagecascade involving Mec1, Rad53, and Dun1, which in turn stimulates higher RNR activity. Importantly, this pool expansion following DNA damage is accompanied by an increased error rate, resulting in thedNTPmutator phenotype (19). Imbalanced ratios of dNTPs are thought to have themostmutagenic potential (22), but proportional increases in dNTP levels can also result in mutagenesis (23). In contrast, significant dNTP pool increases following DNA Proofreading Pol δ/Pol ε Pol Exo