M. Todd Washington

Fidelity and Processivity of Saccharomyces cerevisiae DNA Polymerase η

The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol η, that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA. To better understand the role of pol η in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates. Steady-state kinetic analyses of deoxynucleotide incorporation indicate that pol η has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10−2 to 10−3. Also pol η has a low processivity, incorporating only a few nucleotides before dissociating. We suggest that pol ηs low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors.

Human DINB1-encoded DNA polymerase κ is a promiscuous extender of mispaired primer termini

Both in yeast and humans, DNA polymerase (Pol) η functions in the error-free replication of UV-damaged DNA, and Polη has the unique ability to efficiently replicate through a cis-syn thymine–thymine (T–T) dimer by inserting two As opposite the two Ts of the dimer. Although human DINB1-encoded Polκ belongs to the same protein family as Polη, Polκ shows no ability to bypass this DNA lesion and its biological function has remained unclear. Here, we examine Polκ for its ability to extend from primer-terminal mispairs opposite nondamaged and damaged DNA templates. We find that Polκ is a promiscuous extender of primer-terminal mispairs opposite nondamaged DNA templates, and interestingly, it is also very efficient at extending from a G opposite the 3′T of a T–T dimer. These observations provide biochemical evidence for a role of Polκ in the extension of mismatched base pairs during normal DNA replication, and in addition, they implicate Polκ in the mutagenic bypass of T–T dimers. In its proficient mismatch extension ability, Polκ is more similar to the unrelated DNA polymerase ζ than it is to the phylogenetically related Polη or Polι. Thus, in humans, Polκ would compete with Polζ for the extension of mismatched base pairs on damaged and undamaged DNAs.

Yeast DNA Polymerase η Utilizes an Induced-Fit Mechanism of Nucleotide Incorporation

DNA polymerase eta (Poleta) is unique among eukaryotic DNA polymerases in its proficient ability to replicate through distorting DNA lesions, and Poleta synthesizes DNA with a low fidelity. Here, we use pre-steady-state kinetics to investigate the mechanism of nucleotide incorporation by Poleta and show that it utilizes an induced-fit mechanism to selectively incorporate the correct nucleotide. Poleta discriminates poorly between the correct and incorrect nucleotide at both the initial nucleotide binding step and at the subsequent induced-fit conformational change step, which precedes the chemical step of phosphodiester bond formation. This property enables Poleta to bypass lesions with distorted DNA geometries, and it bestows upon the enzyme a low fidelity.

Efficient and Error-Free Replication Past a Minor-Groove DNA Adduct by the Sequential Action of Human DNA Polymerases ι and κ

ABSTRACT DNA polymerase ι (Polι) is a member of the Y family of DNA polymerases, which promote replication through DNA lesions. The role of Polι in lesion bypass, however, has remained unclear. Polι is highly unusual in that it incorporates nucleotides opposite different template bases with very different efficiencies and fidelities. Since interactions of DNA polymerases with the DNA minor groove provide for the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, we considered the possibility that Polι differs from other DNA polymerases in not being as sensitive to distortions of the minor groove at the site of the incipient base pair and that this enables it to incorporate nucleotides opposite highly distorting minor-groove DNA adducts. To check the validity of this idea, we examined whether Polι could incorporate nucleotides opposite the γ-HOPdG adduct, which is formed from an initial reaction of acrolein with the N2 of guanine. We show here that Polι incorporates a C opposite this adduct with nearly the same efficiency as it does opposite a nonadducted template G residue. The subsequent extension step, however, is performed by Polκ, which efficiently extends from the C incorporated opposite the adduct. Based upon these observations, we suggest that an important biological role of Polι and Polκ is to act sequentially to carry out the efficient and accurate bypass of highly distorting minor-groove DNA adducts of the purine bases.

Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase η

DNA polymerase η (Polη) has the unique ability to replicate through UV-light-induced cyclobutane pyrimidine dimers. Here we use pre-steady-state kinetic analyses to examine the mechanism of nucleotide incorporation opposite a cis-syn thymine-thymine (TT) dimer and an identical nondamaged sequence by yeast Polη. Polη displayed “burst” kinetics for nucleotide incorporation opposite both the damaged and nondamaged templates. Although a slight decrease occurred in the affinity (Kd) of nucleotide binding opposite the TT dimer relative to the nondamaged template, the rate (kpol) of nucleotide incorporation was the same whether the template was damaged or nondamaged. These results strongly support a mechanism in which the nucleotide is directly inserted opposite the TT dimer by using its intrinsic base-pairing ability without any hindrance from the distorted geometry of the lesion.

Efficient and error-free replication past a minor-groove N2-guanine adduct by the sequential action of yeast Rev1 and DNA polymerase ζ

ABSTRACT Rev1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase ζ (Polζ). Rev1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While Rev1 plays an indispensable structural role in Polζ-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, Rev1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N2-adducted guanines in DNA. To test this idea, we examined whether Rev1 could incorporate a C opposite the γ-hydroxy-1,N 2-propano-2′deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N 2 of guanine. Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that Rev1 efficiently incorporates a C opposite this adduct from which Polζ subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the Rev1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N 2-guanine DNA minor-groove adducts that form in DNA.

Accuracy of lesion bypass by yeast and human DNA polymerase η

DNA polymerase η (Polη) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Polη in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Polη replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Polη, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of ≈ 10−2 to 10−3 opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Polη in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Polη to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Polη extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of ≈ 10−2 to 10−3 relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Polη would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3′→5′ exonuclease. Replication through a TT dimer by Polη then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

Variations on a theme: Eukaryotic Y-family DNA polymerases

Most classical DNA polymerases, which function in normal DNA replication and repair, are unable to synthesize DNA opposite damage in the template strand. Thus in order to replicate through sites of DNA damage, cells are equipped with a variety of nonclassical DNA polymerases. These nonclassical polymerases differ from their classical counterparts in at least two important respects. First, nonclassical polymerases are able to efficiently incorporate nucleotides opposite DNA lesions while classical polymerases are generally not. Second, nonclassical polymerases synthesize DNA with a substantially lower fidelity than do classical polymerases. Many nonclassical polymerases are members of the Y-family of DNA polymerases, and this article focuses on the mechanisms of the four eukaryotic members of this family: polymerase eta, polymerase kappa, polymerase iota, and the Rev1 protein. We discuss the mechanisms of these enzymes at the kinetic and structural levels with a particular emphasis on how they accommodate damaged DNA substrates. Work over the last decade has shown that the mechanisms of these nonclassical polymerases are fascinating variations of the mechanism of the classical polymerases. The mechanisms of polymerases eta and kappa represent rather minor variations, while the mechanisms of polymerase iota and the Rev1 protein represent rather major variations. These minor and major variations all accomplish the same goal: they allow the nonclassical polymerases to circumvent the problems posed by the template DNA lesion.

Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase η

ABSTRACT Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase η (Polη) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Polη are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Polη; in this regard, Polη differs strikingly from classical high-fidelity DNA polymerases.

Human DNA Polymerase ι Utilizes Different Nucleotide Incorporation Mechanisms Dependent upon the Template Base

ABSTRACT Human DNA polymerase ι (Polι) is a member of the Y family of DNA polymerases involved in translesion DNA synthesis. Polι is highly unusual in that it possesses a high fidelity on template A, but has an unprecedented low fidelity on template T, preferring to misincorporate a G instead of an A. To understand the mechanisms of nucleotide incorporation opposite different template bases by Polι, we have carried out pre-steady-state kinetic analyses of nucleotide incorporation opposite templates A and T. These analyses have revealed that opposite template A, the correct nucleotide is preferred because it is bound tighter and is incorporated faster than the incorrect nucleotides. Opposite template T, however, the correct and incorrect nucleotides are incorporated at very similar rates, and interestingly, the greater efficiency of G misincorporation relative to A incorporation opposite T arises predominantly from the tighter binding of G. Based on these results, we propose that the incipient base pair is accommodated differently in the active site of Polι dependent upon the template base and that when T is the templating base, Polι accommodates the wobble base pair better than the Watson-Crick base pair.