Standard Article

You have free access to this content

DNA Replication Fidelity

  1. Errol C Friedberg,
  2. Paula L Fischhaber

Published Online: 27 JAN 2006

DOI: 10.1038/npg.els.0006172

eLS

eLS

How to Cite

Friedberg, E. C. and Fischhaber, P. L. 2006. DNA Replication Fidelity. eLS. .

Author Information

  1. UT Southwestern Medical Center, Dallas, Texas, USA

Publication History

  1. Published Online: 27 JAN 2006

Cells are equipped with myriad deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) polymerases that catalyze the formation of phosphodiester bonds between the 3′-hydroxyl of a polynucleotide and the α-phosphate of an incoming nucleoside or deoxynucleo-side 5′-triphosphate (NTP or dNTP). pyrophosphate is eliminated in the course of this reaction. The replicational DNA polymerases achieve copying of DNA molecules in a template-directed fashion, utilizing the opposite DNA strand to direct the correct deoxyribonucleotide for incorporation in order to effect normal Watson–Crick base pairing. Replicational copying of DNA occurs during the S phase of the cell cycle, and the polymerases that operate in this process are called are DNA-directed DNA polymerases.

The intrinsic error rate for any given DNA polymerase is an important feature of DNA replication because uncorrected errors during DNA synthesis lead to the generation of mutations. While a certain level of genetic diversity is crucial for Darwinian evolution, mutagenesis can spell potential disaster for an individual organism by resulting in either hereditary disorders or nonheritable somatic cell diseases such as cancer. It has been estimated that normal cells replicate their genome with a fidelity that translates to approximately a single error per cell generation (Loeb, 1991).

Here we provide an overview of the DNA-directed DNA polymerases and the mechanisms by which the correctly base-paired incoming dNTP is selected for the incorporation of a deoxynucleoside monophosphate residue opposite a template base. Significant discussion is devoted to a consideration of a newly discovered class of DNA polymerases that collectively exhibit low fidelity when copying native DNA, and which appear to have key roles in biologic pathways in which either chemically damaged DNA bases or altered replication forks must be copied to ensure cellular survival, or in which the generation of mutations is physiologically required.

General Features of High-fidelity DNA Polymerases

  1. Top of page
  2. General Features of High-fidelity DNA Polymerases
  3. Biophysical Terms of Replication Fidelity
  4. Low-fidelity DNA Polymerases
  5. Translesion DNA Synthesis
  6. Structural Insights into Low-fidelity Polymerases
  7. Summary
  8. See also
  9. References
  10. Further Reading

Families of DNA polymerases are grouped according to the primary sequence homology of their members and are designated as families A, B, C, D, X, RT and Y. The present discussion will consider classes A and B because these comprise the DNA-directed (DNA-templated) DNA polymerases, including the high-fidelity eukaryotic DNA polymerases. As a general rule, the A family polymerases (e.g. Polγ and Polθ) function in DNA repair, while the B family polymerases (e.g. Polα, Polδ, Polε and Polζ) function primarily in genomic replication. All are believed to synthesize DNA by essentially the same general mechanism. A and B family polymerases contain domains that resemble a side-on view of a right human hand, routinely described as the thumb, fingers and palm domains (Figure 1). They all contain a pocket for the incoming nucleotide (Figure 1), which binds a metal cofactor (Mg2+) that may chelate the incoming nucleotide prior to its binding the polymerase active site.

thumbnail image

Figure 1. Steps of deoxyribonucleic acid (DNA) polymerization depicting the correct incorporation of thymidine 5′-monophosphate. The ‘thumb’, ‘palm’ and ‘finger’ domains are shown in purple, blue and orange respectively. The green domain represents the ‘PAD’ or ‘little finger’ domain that has thus far been identified only in low-fidelity Y family DNA polymerases. The red transluscent domain (in two of the drawings) represents the 3′[RIGHTWARDS ARROW]5′ exonuclease domain, which is not present in Y family polymerases. ‘†’ represents the transition state for enzyme catalysis. dTTP: thymidine 5′-triphosphate; PPi: pyrophosphate; dTMP: thymidine 5′-monophosphate. The curved black arrows denote movement of the ‘thumb’ and ‘fingers’ domains.

DNA polymerases make surprisingly few contacts with the DNA primer-template and the incoming nucleotide, and it is possible to mutate a significant fraction of the amino acids at or near the active site with little or no functional consequence (Patel and Loeb, 2001). Structural studies indicate that DNA polymerases undergo significant conformational shifts between the binding of initial substrates and the binding of a newly synthesized 3′ nucleotide in the 3′[RIGHTWARDS ARROW]5′ exonuclease active site. Only subtle differences in the mechanism between A and B family 3′[RIGHTWARDS ARROW]5′ exonucleolytic proofreading have been identified.

In living cells, DNA polymerases are components of larger holoenzymes and are loaded onto the replication fork by other protein machines known as the clamp and the clamp loader. Other accessory protein factors that are known to modulate polymerase activity and processivity may bear on fidelity to the extent that successive nucleotide insertion events are cooperative. Notably, a recent in vivo study in yeast indicates varying levels of polymerase fidelity on the leading and lagging strands of a DNA replication fork (Pavlov et al., 2002). These caveats are important to bear in mind since mechanistic studies on DNA replication are still largely restricted to in vitro systems that have been accurately reconstituted to varying extents.

Biophysical Terms of Replication Fidelity

  1. Top of page
  2. General Features of High-fidelity DNA Polymerases
  3. Biophysical Terms of Replication Fidelity
  4. Low-fidelity DNA Polymerases
  5. Translesion DNA Synthesis
  6. Structural Insights into Low-fidelity Polymerases
  7. Summary
  8. See also
  9. References
  10. Further Reading

The specifics of the biochemical mechanisms by which any DNA polymerase operates can, in principle, be described mathematically. Numerous studies have been employed to delineate quantitative parameters governing polymerase mechanisms. With respect to DNA polymerase fidelity, the key parameters Vmax, Km and finc can be determined by measuring polymerase action under steady-state kinetics in vitro for each of the four deoxynucleotides and for all possible sequence contexts for nucleotide insertion (Benkovic and Cameron, 1995). Comparisons of the value Vmax/Km for the insertion of each of the four deoxynucleotides at a given primer-template position provide the frequency of misinsertion ( finc). This parameter is described by the equation, finc=Vmax/Km [incorrect]/Vmax/Km [correct]. It is also possible to determine error rates in vivo. This affords a more biologically relevant numerical value but precludes delineation of the relative contributions of dNTP binding (contained in the Km term) and enzymatic catalysis of reactant to product (contained in the Vmax term).

While error rates and other kinetic parameters provide ‘hard’ numbers that can be compared at face value, it is considerably more difficult to delineate the mechanistic origins of observed differences. No fewer than nine distinct models have been proposed for the biochemical mechanism of nucleotide incorporation by DNA polymerase I of E. coli, the best-studied of the high-fidelity replicative polymerases (Beckman and Loeb, 1993). These models share the basic similar features of primer-template binding by the polymerase, subsequent binding of dNTP, phosphodiester bond formation and 3′[RIGHTWARDS ARROW]5′ exonucleolytic proofreading, which excises a mispaired nascent nucleotide in a separate active site located approximately 30–40 Å away (Figure 1).

However, these models differ with regard to several mechanistic steps that bear heavily on the precise mechanism(s) of proofreading and polymerase fidelity. These steps include the possible existence of a nonexonucleolytic proofreading step that may occur at or near the point of phosphodiester bond formation (sometimes referred to as ‘kinetic proofreading’ (Figure 1), the role of solvent exclusion from the active site in correct base selection, the importance of polymerase conformational changes in fidelity during phosphodiester bond formation, details of pyrophosphate release, the connectedness (cooperativity) of the various mechanistic steps, and whether or not the rate-limiting step of the reaction is altered when an incorrect nucleotide is misincorporated.

Detailed protein crystal structures have shed light on several of these conundrums, at least with regard to the particular polymerases studied. It is now clear that two separate conformational changes occur prior to phosphodiester bond formation. As the primer-template binds the polymerase, the ‘thumb’ domain moves inward (Figure 1). Next dNTP binding occurs, accompanied by an inward movement of the ‘fingers’ domain, which brings the soon-to-be formed base pair into alignment (Figure 1). This second conformational change may be as much as 10000-fold slower in the presence of an incorrect dNTP. Additionally, it is believed that the catalysis step itself may be slower in the presence of an incorrect nucleotide resulting from misalignment of the dNTP α-phosphate in the active site.

The last stage of fidelity selection follows phosphodiester bond formation. DNA polymerases harbor 3′[RIGHTWARDS ARROW]5′ exonuclease domains, the location of which relative to the ‘thumb’, ‘fingers’ and ‘palm’ domains varies from one polymerase to another (Figure 1). Crystallographic studies suggest that an incorrectly paired nascent base pair exhibits ‘fraying’ (Figure 1) that favors the partitioning of the 3′ terminus into the exonuclease active site as opposed to remaining in the polymerization active site, thereby favoring nucleotide removal rather than misextension.

Low-fidelity DNA Polymerases

  1. Top of page
  2. General Features of High-fidelity DNA Polymerases
  3. Biophysical Terms of Replication Fidelity
  4. Low-fidelity DNA Polymerases
  5. Translesion DNA Synthesis
  6. Structural Insights into Low-fidelity Polymerases
  7. Summary
  8. See also
  9. References
  10. Further Reading

In recent years, a large number of related genes have been recognized to code for a new class of DNA polymerases that are represented in species ranging from E. coli to humans (Ohmori et al., 2001). These polymerases lack 3′[RIGHTWARDS ARROW]5′ proofreading exonuclease activity and share the additional properties of low fidelity when copying undamaged DNA, as well as low processivity, incorporating only a few nucleotides prior to dissociation from the primer-template. Additionally, the Y family members exhibit the ability to support DNA synthesis in vitro across and beyond a variety of DNA lesions that typically arrest high-fidelity replicative polymerases of the A and B families in eukaryotes. In humans, polymerases η, ι and κ are all Y family members. A number of other newly discovered polymerases that are not members of the Y family also exhibit low fidelity and, in at least one case, the ability to bypass certain forms of DNA damage. Notable examples include the human polymerases λ, μ and ζ. The number of known polymerases in humans is now 14, up from five just four years ago.

In the light of the requirement for extremely high accuracy in copying the genome to ensure the fidelity of genetic inheritance, the existence of a large number of polymerases with low fidelity that are additionally devoid of exonucleolytic proofreading capability may appear paradoxical. However, the Y family members appear to subserve a critical and specific role in promoting cellular survival in the presence of template base damage that arrests or impedes high-fidelity DNA replication. This function is referred to as DNA lesion bypass or translesion DNA synthesis (Prakash and Prakash, 2002). Additional physiologic mutagenic roles for these polymerases have been proposed, specifically during the process of somatic hypermutation in immunoglobulin genes (Gearhart and Wood, 2001). Some of the Y family polymerases are also expressed preferentially in specific cells in the testis, suggesting a possible role in meiosis or some other aspect of germ cell differentiation.

Translesion DNA Synthesis

  1. Top of page
  2. General Features of High-fidelity DNA Polymerases
  3. Biophysical Terms of Replication Fidelity
  4. Low-fidelity DNA Polymerases
  5. Translesion DNA Synthesis
  6. Structural Insights into Low-fidelity Polymerases
  7. Summary
  8. See also
  9. References
  10. Further Reading

One of the most extensively biologically and biochemically characterized members of the Y family is the enzyme DNA polymerase eta (Polη). This enzyme supports translesion synthesis across a spectrum of noncoding base damage in DNA, including cyclobutane pyrimidine dimers, a form of ultraviolet radiation-induced DNA damage that is potently arresting both to DNA and RNA polymerases. Polη synthesizes DNA largely correctly past cyclobutane pyrimidine dimers, inserting adenine in preference to other bases opposite each of the two thymines in a thymine dimer with an efficiency of over 99%.

Importantly, the ability of Polη to bypass thymine dimers accurately in vitro is supported by biologic studies. Mutations in the gene for Polη (XPV) can result in a hereditary disease known as xeroderma pigmentosum, which is characterized by extreme sensitivity to sunlight and a profound predisposition to skin cancer at a very early age. This observation has led to the cogent hypothesis that in normal individuals, thymine (and possibly other pyrimidine) dimers that are active sites of replicative arrest are efficiently and accurately bypassed by Polη, thereby preventing both cell death and mutations. In the absence of Polη, one or more other low-fidelity polymerases subserve the bypass function critical for cell survival, but with a high probability of mutation. The mutational burden so generated eventually leads to sunlight-induced skin cancer.

Individual members of the Y family of polymerases do not bypass identical arrays of DNA lesions, suggesting that each polymerase may be preferred for a different subset of lesions generated by spontaneous and naturally occurring environmental sources of DNA damage. The many DNA lesions routinely observed in cells may explain the existence of such a large number of DNA polymerases in eukaryotic cells.

Structural Insights into Low-fidelity Polymerases

  1. Top of page
  2. General Features of High-fidelity DNA Polymerases
  3. Biophysical Terms of Replication Fidelity
  4. Low-fidelity DNA Polymerases
  5. Translesion DNA Synthesis
  6. Structural Insights into Low-fidelity Polymerases
  7. Summary
  8. See also
  9. References
  10. Further Reading

Several recent X-ray crystal structures have demonstrated that the active site of the Y family members possess a relatively open, solvent-accessible binding pocket for the template base and the incoming dNTP (Friedberg et al., 2001). Solvent accessibility to the active site may be key in the light of the newfound ability to compare Y family polymerase X-ray structures with those of high-fidelity DNA polymerases such as Polα, which show a more closed configuration at the active site. Solvent molecules can hydrogen bond to the base of the incoming dNTP, which is expected to lower the free energy difference between binding the correct dNTP and incorrect dNTPs. Hence, exclusion of solvent from the active site can maximize polymerase discrimination for the correct base. The different solvent accessibilities between high-fidelity and Y family DNA polymerases may be indicative of the importance of solvent exclusion for the binding of the correct dNTP and may explain the lowered discrimination for the correct nucleotide by Y family members. Finally, work with DNA base analogs lacking the hydrogen bonding moieties of the standard bases has revealed that correct base binding depends largely on the geometry of the incoming base. It has been proposed that base solvation makes the base geometrically larger and that failure to exchange solvent molecules for contacts with the template base prevents an incorrect base from fitting into the active site.

Lack of 3′[RIGHTWARDS ARROW]5′ exonucleolytic proofreading activity is expected to reduce the overall fidelity of a DNA polymerase by about 10-fold. Clearly, this parameter alone cannot explain the 100–1000 (or more) reduced overall fidelity of the Y family members compared to the high-fidelity polymerase families (Friedberg et al., 2002). Hence, it will be interesting to determine which other parameters contribute to overall lowered fidelity through biochemical and biophysical probing of the Y family members. In particular, does the additional 10- to 100-fold reduction in fidelity arise exclusively from increased solvent accessibility to the active site or by other subtle alterations in polymerase mechanism(s)?

Summary

  1. Top of page
  2. General Features of High-fidelity DNA Polymerases
  3. Biophysical Terms of Replication Fidelity
  4. Low-fidelity DNA Polymerases
  5. Translesion DNA Synthesis
  6. Structural Insights into Low-fidelity Polymerases
  7. Summary
  8. See also
  9. References
  10. Further Reading

Replicational fidelity is crucial for cellular survival, evolution and the prevention of disease. Cells are not driven merely to acquiring the most accurate possible copy of their DNA. There is also biologic pressure toward generating mutations that can provide the essential genetic diversity for selection during Darwinian evolution. Additionally, cells require special DNA copying circumstances, including the bypass of base damage and possibly the generation of mutations to enhance immunologic diversity. The origins of polymerase fidelity are complicated, and it is clear that this important class of enzymes employ more than a single mechanism to achieve the level of accuracy needed for their distinct biochemical roles, be that highly accurate genome copying, DNA lesion bypass, gap-filling during DNA repair or immunologic diversification during somatic hypermutation.

References

  1. Top of page
  2. General Features of High-fidelity DNA Polymerases
  3. Biophysical Terms of Replication Fidelity
  4. Low-fidelity DNA Polymerases
  5. Translesion DNA Synthesis
  6. Structural Insights into Low-fidelity Polymerases
  7. Summary
  8. See also
  9. References
  10. Further Reading

Further Reading

  1. Top of page
  2. General Features of High-fidelity DNA Polymerases
  3. Biophysical Terms of Replication Fidelity
  4. Low-fidelity DNA Polymerases
  5. Translesion DNA Synthesis
  6. Structural Insights into Low-fidelity Polymerases
  7. Summary
  8. See also
  9. References
  10. Further Reading