Mutator phenotypes due to DNA replication infidelity

2.1. The major replicative DNA polymerases

Pols α, δ and ε perform the vast majority of nuclear DNA replication. Several models have been put forth for their division of labor at the nuclear DNA replication fork [5,6]. These models all share the idea that initiation of replication at origins and of each Okazaki fragment on the lagging strand template requires the RNA primase activity associated with Pol α to synthesize RNA primers of ~10 bases (Fig. 2A). Pol α then extends these primers by incorporating about 20–30 deoxynucleotides. Once DNA chains are initiated, current evidence [5,7] suggests that the leading strand template may be copied primarily by Pol ε, a highly processive enzyme with an associated proofreading exonuclease activity. Pol δ copies primarily the lagging strand template, generating a series of Okazaki fragments of approximately 200–300 base pairs each that are ultimately processed to remove RNA primers and permit ligation [8].

2.2. The TLS polymerases

Although the above situation may apply to replicating undamaged DNA, enzymology becomes more complicated when the fork encounters lesions that distort the DNA helix (Fig. 2B). Family B members like Pols α, δ and ε depend heavily on normal helix geometry for efficient and accurate synthesis [1,9], such that helix distorting lesions can slow or prevent replication fork progression. To solve this problem, a stalled replication fork can elicit one or more switches among DNA polymerases capable of bypassing lesions. The specialized TLS polymerases include Pols ζ, η, κ, ι and Rev1 (Y family) and Pols θ and ν (A family). These polymerases can incorporate nucleotides opposite lesions and/or extend the resulting primer terminus for one or more nucleotides, thus creating primer-templates that can be used by the major replication enzymes [10,11]. The number of switches involved in TLS, and the identity and number of polymerases required, can vary depending on the lesion, of which there are many types that differ by composition and structure [12]. The number of switches also depends on the substrate preferences of the various polymerases, which also vary widely and can partially overlap. The location and timing of TLS may vary, sometimes occurring at the fork during ongoing DNA replication, sometimes occurring during post-replication gapfilling synthesis, e.g., catalyzed by Pol ζ [13], or perhaps occurring during excision repair (e.g., Pol κ). In fact, eukaryotic DNA polymerases other than those listed in Table 1 also have been implicated in TLS, including two family X members Pol β and Pol λ that fill short gaps during base excision repair and non-homologous end joining of double strand breaks in DNA. Pol β has been shown to be involved with bypass of an abasic site and a d(GpG)-cisplatin adduct [14,15] and PCNA was found to efficiently stimulate the bypass of an abasic site by Pol λ [2,16,17]. Readers interested in the substrate specificities of the TLS polymerases and mechanisms of polymerase switching involving post-translational modifications of polymerases and their accessory proteins can consult any of several recent reviews on this topic [4,18,19].

3.1. Nucleotide selectivity/error prevention

Most DNA polymerases lack intrinsic 3′-exonuclease activity to excise errors. Therefore, their fidelity depends on the ability to prevent incorporation of incorrect dNTPs that lead to base substitutions, and to prevent incorporation involving misaligned substrates that leads to loss or addition of nucleotides. Amazingly, DNA polymerase selectivity can vary over a million-fold range (Fig. 1).

3.2. Proofreading

Once an incorrect dNTP is incorporated into DNA, the mismatched primer terminus is more difficult to extend than a correctly paired and properly aligned primer terminus. The delay in extension caused by a mismatch allows the primer terminus to fray and travel (switch) from the polymerase to the exonuclease active site for excision of the error [35].

5.1. Reduced nucleotide selectivity

Nucleotide selectivity can be reduced by mutations in polymerase genes that alter amino acids comprising the active site or the nascent base pair binding pocket, or even more distant amino acids that indirectly influence selectivity [42] (for further review please see chapter by Sweasy). For the major replicative polymerases whose polymerase activities are critical for cell viability, the probability that such defects will be associated with cancer is likely to be small, because most such mutations reduce polymerase activity and therefore reduce proliferative potential. The probability that a dNTP will be misinserted also depends on the relative concentrations of each of the four dNTPs available for DNA synthesis. For example, an abnormally high concentration of dTTP relative to dCTP will promote misinsertion of dTMP opposite template guanine which, if uncorrected, will yield G–C to T–A substitutions. Interestingly, the four dNTP concentrations are not equal even in normal cells (e.g., the dGTP concentration is typically lower than the other three), and they are highly regulated throughout the cell cycle as well as in response to environmental stress. Any deviation from the normal dNTP ratios has the potential to decrease replication fidelity and generate a mutator phenotype. This is illustrated by the elevated mutation rates in cells containing mutations in genes that regulate dNTP concentrations, e.g., ribonucleotide reductase [43–45]. dNTP pool imbalances induced by means other than genetic defects, e.g., by environmental stress, also have the potential to elevate mutation rates. This could occur in a transient manner that would contribute only to a subset of the multiple mutations needed for tumor formation. dNTP pool imbalances that result from increasing one or more dNTP concentrations may be more relevant to cancer than imbalances resulting from decreasing dNTP concentrations below normal levels, because the latter can limit proliferative potential [44].

5.2. Reduced proofreading efficiency

The efficiency of proofreading depends on a balance between polymerization from terminal mismatches and 3′-exonucleolytic excision of the error. Thus, defects to either activity can reduce the contribution of proofreading to replication fidelity (Fig. 1). The exonuclease can be inactivated by active site mutations. It can be reduced by the presence of dNMPs, which are the end products of excision that can bind to the exonuclease active site and inhibit activity. Proofreading can be reduced by mutations that alter the ability of the primer terminus to switch from the polymerase to the exonuclease active site. Proofreading can be suppressed by mutations in the polymerase active site that promote mismatch extension, or by dNTP pool changes that increase the concentration of the correct dNTPs needed to extend mismatches.

Proofreading potential can also be affected by DNA damage. A fine example involves the common oxidative lesion, 8-oxo-guanine. In its syn conformation, 8-oxo-G pairs with adenine and largely escapes proofreading because it mimics the shape of a correct base pair within the nascent base pair binding pocket of the polymerase. Finally, even some undamaged mismatches are simply poor substrates for normal proofreading. The best example involves inefficient proofreading of insertion and deletion mismatches in repetitive sequences. Efficiency decreases as the length of a repetitive sequence increases, because the unpaired base can reside upstream of the terminus, where it can be protected from excision by intervening correct base pairs [21]. In principle, any of these mechanisms could be relevant to a mutator phenotype associated with cancer, because proofreading is dispensable for eukaryotic cell viability.

5.3. Reduced MMR efficiency

MMR is also dispensable for eukaryotic cell viability, yet critical for replication fidelity. Like proofreading, MMR efficiency can be reduced by mutations in genes whose products are required for MMR (Fig. 1). The degree to which these genetic defects elevate mutation rates varies considerably, depending on the nature of the gene mutation, the function of the particular gene product in MMR and the type and location of the replication error considered. Another way to reduce MMR efficiency is to change the expression of MMR genes. MMR can be inactivated by promoter hypermethylation to silence expression of the essential human MMR gene MLH1, a mechanism strongly associated with spontaneous colon cancer. The level of MMR proteins needed to correct replication errors under normal conditions may be insufficient when replication errors accumulate at a higher rate. For example, studies have shown that MMR capacity can be saturated by rapid error-prone replication in Escherichia coli, or by the presence of DNA lesions to which MMR proteins can bind. In yeast, over-expression of two MMR genes, MLH1 and MSH3, reduces MMR efficiency, presumably by perturbing normal protein–protein partnerships required for MMR. Exposure of budding yeast to cadmium, a metal that is ubiquitously present in the environment and is a known human carcinogen, can inhibit MMR and strongly elevate the mutation rate [46]. There potentially may be other environmental exposures that inhibit MMR. Reduced MMR due to altered expression, saturation, or cadmium inhibition is reversible; again potentially generating transient mutator phenotypes.

7.1. Microsatellite instability

A highly successful current example is microsatellite instability (MSI), the loss or gain of base pairs in short (1–6 base) repetitive DNA sequences. These mutations result from strand slippage during replication to generate one or more unpaired bases. When slippage occurs in repetitive sequences, the extra base(s) comprising the mismatch can be present in otherwise duplex DNA, well upstream of the primer terminus and stabilized by intervening correct base pairs whose numbers increase as the number of base pairs in the repetitive sequence increase [57–59]. As a consequence, slipped mismatch intermediates are not only frequently generated during replication, they are also more readily extended than terminal mismatches and therefore inefficiently proofread. For these reasons, MMR is the major guardian of genome stability against MSI, as illustrated by the observation that a MMR defect can increase the rate of certain single base deletions by 100,000-fold [60,61]. This is why MSI is now a widely used “replication error signature” that is diagnostic of MMR defective tumors [62–66] (for further review see chapter by Salk and Horwitz). MSI may be a useful biomarker for causes of mutator phenotypes other than MMR defects. For example, a recent study reported that if ribonucleotides incorporated into DNA during replication by yeast Pol ε are not removed by RNase H2-dependent repair, they result in 2–5 base pair deletions in repetitive sequences [67].

7.2. Somatic hypermutation

Another clear example of a useful error signature, albeit one not directly related to cancer per se, involves a base substitution signature for somatic hypermutation (SHM) of immunoglobulin genes. SHM generates mutations in the variable regions of immunoglobulin genes at an extraordinary rate, thereby contributing to affinity maturation of antibodies [68]. Initial studies of the error specificity of Pol η in vitro demonstrated that human and mouse Pol η preferentially generate mismatches at A–T base pairs, and preferentially in certain sequence contexts [69]. This motivated subsequent studies (e.g. [70–73]) that now strongly support the idea that Pol η is indeed responsible for much of the SHM occurring in vivo at A–T base pairs. Table 2 lists other point mutation signatures of replication infidelity that eventually may have diagnostic relevance for mutator phenotypes and cancer.

7.3. Tandem double base substitutions

Forming a tandem double base substitution by DNA synthesis requires misinsertion, followed by another misinsertion onto a mismatched terminus, and then followed by multiple extensions of a doubly mismatched terminus. Each of these steps alone is normally rare, and therefore accurate replicative DNA polymerases have rarely been seen to generate tandem double base substitutions. However, Pol κ [74], Pol ζ [75] and especially Pol η [69,72,76], generate tandem double base substitutions at readily detectable rates when copying undamaged DNA. In fact, the accuracy of Pol η is so low that it also generates tandem triple substitutions and mutants with substitutions separated by 1–3 base pairs. Tandem double base substitutions are also a characteristic feature of mutagenesis resulting from exposure to UV light, e.g., CC to TT mutations that are likely generated during TLS of UU dimers resulting from deamination of CC dimers. For these reasons, tandem double base substitutions are a biomarker for involvement of TLS polymerases.

7.4. Clustered-complex and multiple mutations generated by Pol ζ

When copying undamaged DNA in vitro, Pol ζ generates multiple substitutions, deletions and/or insertions within a few base pairs of each other. Pol ζ also generates such complex errors in vivo [77,78], more so in genetic backgrounds defective in processing DNA damage. Highly mutagenic DNA synthesis by yeast Pol ζ also has recently been observed when copying long tracts of damaged single stranded DNA [79]. Such long tracts can be generated during the end-resection phase of DNA double strand break repair [80], or by uncoupling leading and lagging strand replication. This Pol ζ-dependent mutagenesis can introduce multiple mutations spread over thousands of base pairs. Whether tandem, clustered or spread out, the concerted introduction of multiple mutations in a single DNA transaction provides an opportunity for selection in tumor progression, or in evolution, that differs from the selective advantages of single point mutations introduced in series over multiple cell generations (for further discussion, see [79,81,82] and references therein).

7.5. Unusual error signatures of other DNA polymerases

Other TLS polymerases also have unusual error specificities (Table 2). These include Pol ι, which frequently inserts incorrect dNTPs opposite template pyrimidines [83,84], Pol ν, which forms T-dGTP mismatches at an unusually high rate [85,86], and Pol θ, which generates single base additions at a higher rate than other polymerases [87]. In addition, mutant alleles of the three major replicative polymerases have high error rates for some mismatches but not others. These biases have been useful for assigning the roles of Pol δ and Pol ε in replicating the leading and lagging strand templates in yeast [7,8], and for identifying potential mutable sequence motifs for Pol δ errors in a whole yeast genome sequencing study [88].

The above examples of replication error specificity illustrate the potential value of testing the mutator hypothesis by not only looking for increases in spontaneous and stress-induced mutation rates and frequencies, but also actually using replication error signatures as clues to the molecular nature of the defects that initiate and/or promote tumor formation.