DNA Polymerase β Ribonucleotide Discrimination: INSERTION,MISINSERTION, EXTENSION,AND CODING*

DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct (deoxyribose) sugar. Sugar discrimination represents a great challenge because ribonucleotide triphosphates are present at much higher cellular concentrations than their deoxy-counterparts. Although DNA polymerases discriminate against ribonucleotides, many therapeutic nucleotide analogs that target polymerases have sugar modifications, and their efficacy depends on their ability to be incorporated into DNA. Here, we investigate the ability of DNA polymerase β to utilize nucleotides with modified sugars. DNA polymerase β readily inserts dideoxynucleoside triphosphates but inserts ribonucleotides nearly 4 orders of magnitude less efficiently than natural deoxynucleotides. The efficiency of ribonucleotide insertion is similar to that reported for other DNA polymerases. The poor polymerase-dependent insertion represents a key step in discriminating against ribonucleotides because, once inserted, a ribonucleotide is easily extended. Likewise, a templating ribonucleotide has little effect on insertion efficiency or fidelity. In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3′-endo sugar pucker facilitates nucleotide insertion, whereas a 2′-endo conformation inhibits insertion.     

Sphingosine,a Modulator of Human Translesion DNA Polymerase Activity

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼3000 small molecules, including one comprising ∼600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.     

Frequency and patterns of ribonucleotide incorporation around autonomously replicating sequences in yeast reveal the division of labor of replicative DNA polymerases

Ribonucleoside triphosphate (rNTP) incorporation in DNA by DNA polymerases is a frequent phenomenon that results in DNA structural change and genome instability. However, it is unclear whether the rNTP incorporation into DNA follows any specific sequence patterns. We analyzed multiple datasets of ribonucleoside monophosphates (rNMPs) embedded in DNA, generated from three rNMP-sequencing techniques. These rNMP libraries were obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late-firing autonomously replicating sequences (ARSs) of the yeast genome, where leading and lagging DNA synthesis starts bidirectionally. We found the preference of rNTP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNTP incorporation changes dramatically within the first 1,000 nucleotides from ARSs, highlighting the Pol δ - Pol ϵ handoff during early leading-strand synthesis. Furthermore, the pattern of rNTP incorporation is markedly distinct between the leading and lagging strands not only in mutant but also in wild-type polymerase cells. Such specific signatures of Pol δ and Pol ϵ provide a new approach to track the labor of these polymerases.     

Enzymatic switching for efficient and accurate translesion DNA replication

When cyclobutane pyrimidine dimers stall DNA replication by DNA polymerase (Pol) δ or ε, a switch occurs to allow translesion synthesis by DNA polymerase η, followed by another switch that allows normal replication to resume. In the present study, we investigate these switches using Saccharomyces cerevisiae Pol δ, Pol ε and Pol η and a series of matched and mismatched primer templates that mimic each incorporation needed to completely bypass a cis–syn thymine–thymine (TT) dimer. We report a complementary pattern of substrate use indicating that enzymatic switching involving localized translesion synthesis by Pol η and mismatch excision and polymerization by a major replicative polymerase can account for the efficient and accurate dimer bypass known to suppress sunlight-induced mutagenesis and skin cancer.     

Response of human DNA polymerase iota to DNA lesions

Lesion bypass is an important mechanism to overcome replication blockage by DNA damage. Translesion synthesis requires a DNA polymerase (Pol). Human Pol ι encoded by the RAD30B gene is a recently identified DNA polymerase that shares sequence similarity to Pol η. To investigate whether human Pol ι plays a role in lesion bypass we examined the response of this polymerase to several types of DNA damage in vitro. Surprisingly, 8-oxoguanine significantly blocked human Pol ι. Nevertheless, translesion DNA synthesis opposite 8-oxoguanine was observed with increasing concentrations of purified human Pol ι, resulting in predominant C and less frequent A incorporation opposite the lesion. Opposite a template abasic site human Pol ι efficiently incorporated a G, less frequently a T and even less frequently an A. Opposite an AAF-adducted guanine, human Pol ι was able to incorporate predominantly a C. In both cases, however, further DNA synthesis was not observed. Purified human Pol ι responded to a template TT (6–4) photoproduct by inserting predominantly an A opposite the 3′ T of the lesion before aborting DNA synthesis. In contrast, human Pol ι was largely unresponsive to a template TT cis-syn cyclobutane dimer. These results suggest a role for human Pol ι in DNA lesion bypass.     

Strand displacement synthesis by yeast DNA polymerase ε

DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3′–5′ exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3′–5′ exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5′ end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair.     

The efficiency and fidelity of 8-oxo-guanine bypass by DNA polymerases δ and η

A DNA lesion created by oxidative stress is 7,8-dihydro-8-oxo-guanine (8-oxoG). Because 8-oxoG can mispair with adenine during DNA synthesis, it is of interest to understand the efficiency and fidelity of 8-oxoG bypass by DNA polymerases. We quantify bypass parameters for two DNA polymerases implicated in 8-oxoG bypass, Pols δ and η. Yeast Pol δ and yeast Pol η both bypass 8-oxoG and misincorporate adenine during bypass. However, yeast Pol η is 10-fold more efficient than Pol δ, and following bypass Pol η switches to less processive synthesis, similar to that observed during bypass of a cis-syn thymine-thymine dimer. Moreover, yeast Pol η is at least 10-fold more accurate than yeast Pol δ during 8-oxoG bypass. These differences are maintained in the presence of the accessory proteins RFC, PCNA and RPA and are consistent with the established role of Pol η in suppressing ogg1-dependent mutagenesis in yeast. Surprisingly different results are obtained with human and mouse Pol η. Both mammalian enzymes bypass 8-oxoG efficiently, but they do so less processively, without a switch point and with much lower fidelity than yeast Pol η. The fact that yeast and mammalian Pol η have intrinsically different catalytic properties has potential biological implications.     

DNA polymerases ζ and Rev1 mediate error-prone bypass of non-B DNA structures

DNA polymerase ζ (Pol ζ) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ζ can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ζ in handling these disturbances. We show that Pol ζ/Rev1-dependent mutations occur at sites of replication stalling at short repeated sequences capable of forming hairpin structures. The Rev1 deoxycytidyl transferase can take over the stalled replicative polymerase and incorporate an additional ‘C’ at the hairpin base. Full hairpin bypass often involves template-switching DNA synthesis, subsequent realignment generating multiply mismatched primer termini and extension of these termini by Pol ζ. The postreplicative pathway dependent on polyubiquitylation of proliferating cell nuclear antigen provides a backup mechanism for accurate bypass of these sequences that is primarily used when the Pol ζ/Rev1-dependent pathway is inactive. The results emphasize the pivotal role of noncanonical DNA structures in mutagenesis and reveal the long-sought-after mechanism of complex mutations that represent a unique signature of Pol ζ.     

Switching between polymerase and exonuclease sites in DNA polymerase ε

The balance between exonuclease and polymerase activities promotes DNA synthesis over degradation when nucleotides are correctly added to the new strand by replicative B-family polymerases. Misincorporations shift the balance toward the exonuclease site, and the balance tips back in favor of DNA synthesis when the incorrect nucleotides have been removed. Most B-family DNA polymerases have an extended β-hairpin loop that appears to be important for switching from the exonuclease site to the polymerase site, a process that affects fidelity of the DNA polymerase. Here, we show that DNA polymerase ε can switch between the polymerase site and exonuclease site in a processive manner despite the absence of an extended β-hairpin loop. K967 and R988 are two conserved amino acids in the palm and thumb domain that interact with bases on the primer strand in the minor groove at positions n−2 and n−4/n−5, respectively. DNA polymerase ε depends on both K967 and R988 to stabilize the 3′-terminus of the DNA within the polymerase site and on R988 to processively switch between the exonuclease and polymerase sites. Based on a structural alignment with DNA polymerase δ, we propose that arginines corresponding to R988 might have a similar function in other B-family polymerases.     

Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase

Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 10⁵, fidelity is further enhanced several orders of magnitude by a 3′–5′ proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.     

Structural basis of DNA polymerase θ mediated DNA end joining

DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes microhomologous DNA ends and performs low-fidelity DNA synthesis remain unclear. Here, we present cryo-electron microscope structures of the polymerase domain of Lates calcarifer Pol θ with long and short duplex DNA at up to 2.4 Å resolution. Interestingly, Pol θ binds to long and short DNA substrates similarly, with extensive interactions around the active site. Moreover, Pol θ shares a similar active site as high-fidelity A-family polymerases with its finger domain well-closed but differs in having hydrophilic residues surrounding the nascent base pair. Computational simulations and mutagenesis studies suggest that the unique insertion loops of Pol θ help to stabilize short DNA binding and assemble the active site for MMEJ repair. Taken together, our results illustrate the structural basis of Pol θ-mediated MMEJ.     

Solution structures of 2 : 1 and 1 : 1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle X-ray scattering

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme–DNA complexes of rat DNA polymerase β (Pol β) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol β–DNA complex, whereas only 1 : 1 Pol X–DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol β–DNA and 1 : 1 Pol X–DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5′-dRP lyase domain of the second Pol β molecule with the active site of the 1 : 1 Pol β–DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5′-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5′-dRP lyase reaction in the crystal structures of the closed ternary enzyme–DNA–dNTP complexes, we propose that the asymmetric 2 : 1 Pol β–DNA complex enhances the function of Pol β.     

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol ζ is substantially lower than that of homologous B family pols α, δ and . Pol ζ is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.     

Exchange between Escherichia coli polymerases II and III on a processivity clamp

Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, β. Single-molecule experiments reveal that the interactions of Pol II and Pol III with β allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a β-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork.     

Promiscuous mismatch extension by human DNA polymerase lambda

DNA polymerase lambda (Pol λ) is one of several DNA polymerases suggested to participate in base excision repair (BER), in repair of broken DNA ends and in translesion synthesis. It has been proposed that the nature of the DNA intermediates partly determines which polymerase is used for a particular repair reaction. To test this hypothesis, here we examine the ability of human Pol λ to extend mismatched primer-termini, either on ‘open’ template-primer substrates, or on its preferred substrate, a 1 nt gapped-DNA molecule having a 5′-phosphate. Interestingly, Pol λ extended mismatches with an average efficiency of ≈10⁻² relative to matched base pairs. The match and mismatch extension catalytic efficiencies obtained on gapped molecules were ≈260-fold higher than on template-primer molecules. A crystal structure of Pol λ in complex with a single-nucleotide gap containing a dG·dGMP mismatch at the primer-terminus (2.40 Å) suggests that, at least for certain mispairs, Pol λ is unable to differentiate between matched and mismatched termini during the DNA binding step, thus accounting for the relatively high efficiency of mismatch extension. This property of Pol λ suggests a potential role as a ‘mismatch extender’ during non-homologous end joining (NHEJ), and possibly during translesion synthesis.     

3D architecture of DNA Pol α reveals the functional core of multi-subunit replicative polymerases

Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol α, Pol δ and Pol . The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol α. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol α reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B–CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases.     

DNA polymerase η promotes nonhomologous end joining upon etoposide exposure dependent on the scaffolding protein Kap1

DNA polymerase eta (Pol η) is a eukaryotic member of the Y-family of DNA polymerase involved in translesion DNA synthesis and genome mutagenesis. Recently, several translesion DNA synthesis polymerases have been found to function in repair of DNA double-strand breaks (DSBs). However, the role of Pol η in promoting DSB repair remains to be well defined. Here, we demonstrated that Pol η could be targeted to etoposide (ETO)-induced DSBs and that depletion of Pol η in cells causes increased sensitivity to ETO. Intriguingly, depletion of Pol η also led to a nonhomologous end joining repair defect in a catalytic activity–independent manner. We further identified the scaffold protein Kap1 as a novel interacting partner of Pol η, the depletion of which resulted in impaired formation of Pol η and Rad18 foci after ETO treatment. Additionally, overexpression of Kap1 failed to restore Pol η focus formation in Rad18-deficient cells after ETO treatment. Interestingly, we also found that Kap1 bound to Rad18 in a Pol η-dependent manner, and moreover, depletion of Kap1 led to a significant reduction in Rad18–Pol η association, indicating that Kap1 forms a ternary complex with Rad18 and Pol η to stabilize Rad18–Pol η association. Our findings demonstrate that Kap1 could regulate the role of Pol η in ETO-induced DSB repair via facilitating Rad18 recruitment and stabilizing Rad18–Pol η association.     

A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases

Specialized DNA polymerases are involved in DNA synthesis during base-excision repair and translesion synthesis across a wide range of chemically modified DNA templates. Notable features of these enzymes include low catalytic efficiency, low processivity and low fidelity. Traditionally, in vitro studies of these enzymes have utilized radiolabeled substrates and gel electrophoretic separation of products. We have developed a simple homogeneous fluorescence-based method to study the enzymology of specialized DNA polymerases in real time. The method is based on fluorescent reporter strand displacement from a tripartite substrate containing a quencher-labeled template strand, an unlabeled primer and a fluorophore-labeled reporter. With this method, we could follow the activity of human DNA polymerases β, η, ι and κ under different reaction conditions, and we investigated incorporation of the aberrant nucleotide, 8-oxodGTP, as well as bypass of an abasic site or 8-oxoG DNA template lesion in different configurations. Lastly, we demonstrate that the method can be used for small molecule inhibitor discovery and characterization in highly miniaturized settings, and we report the first nanomolar inhibitors of Y-family DNA polymerases ι and η. The fluorogenic method presented here should facilitate mechanistic and inhibitor investigations of these polymerases and is also applicable to the study of highly processive replicative polymerases.     

Structural basis for inhibition of DNA replication by aphidicolin

Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.