Structure of eukaryotic DNA polymerase δ bound to the PCNA clamp while encircling DNA.

The DNA polymerase (Pol) δ of Saccharomyces cerevisiae (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Šcryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to "waterskate" along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to Escherichia coli replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.

Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination.

The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.

CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels.

In this study, we report that the tetraspanin CD81 enhances human immunodeficiency virus (HIV)-1 reverse transcription in HIV-1-infected cells. This is enabled by the direct interaction of CD81 with the deoxynucleoside triphosphate phosphohydrolase SAMHD1. This interaction prevents endosomal accumulation and favours the proteasome-dependent degradation of SAMHD1. Consequently, CD81 depletion results in SAMHD1 increased expression, decreasing the availability of deoxynucleoside triphosphates (dNTP) and thus HIV-1 reverse transcription. Conversely, CD81 overexpression, but not the expression of a CD81 carboxy (C)-terminal deletion mutant, increases cellular dNTP content and HIV-1 reverse transcription. Our results demonstrate that the interaction of CD81 with SAMHD1 controls the metabolic rate of HIV-1 replication by tuning the availability of building blocks for reverse transcription, namely dNTPs. Together with its role in HIV-1 entry and budding into host cells, the data herein indicate that HIV-1 uses CD81 as a rheostat that controls different stages of the infection.

A CRISPR/Cas9 approach reveals that the polymerase activity of DNA polymerase ß is dispensable for HIV-1 infection in dividing and nondividing cells.

Retrovirus integration into the host genome relies on several host enzymes, potentially including DNA polymerase ß (Pol ß). However, whether human Pol ß is essential for lentivirus replication in human cells is unclear. Here, we abolished DNA polymerase ß (Pol ß) expression by targeting its DNA polymerase domain with CRISPR/Cas9 in human monocytic THP-1 cells to investigate the role of Pol ß in HIV-1 transduction in both dividing and nondividing macrophage stages of THP-1 cells. Pol ß-knock-out was confirmed by enhanced sensitivity to methyl methanesulfonate-induced DNA damage. Of note, nuclear extracts from Pol ß-knock-out THP-1 cells prepared from both dividing and nondividing stages displayed significantly reduced capability to repair the gapped HIV-1 integration intermediate DNA substrate in a biochemical simulation. However, nuclear extract from both dividing and nondividing stages of the Pol ß-KO cells had detectable gap repair activity, suggesting that other host DNA polymerases also repair gapped HIV-1 DNA, particularly in dividing cells. Next, when we compared transduction using HIV-1 and simian immunodeficiency virus in control and Pol ß-KO cells, the loss of the Pol ß expression did not affect transduction efficiency of these lentiviruses in both dividing and nondividing stages. Finally, the gap repair assay indicated that limited cellular dNTP pools, but not Pol ß expression, are a primary factor for HIV-1 DNA gap repair, particularly in nondividing cells. These data support the idea that Pol ß polymerase activity is dispensable for HIV-1 infection in both dividing and nondividing stages of human cells targeted by the virus.

Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol.

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn2+-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn2+, rather than Mg2+, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn2+ and Mg2+, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn2+ than with Mg2+. Notably, the substitution fidelity of PrimPol in the presence of Mn2+ was determined to be in the range of 3.4×10-2 to 3.8×10-1, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn2+ and 150-4500 with Mg2+, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).

New nucleotide-competitive non-nucleoside inhibitors of terminal deoxynucleotidyl transferase: discovery, characterization, and crystal structure in complex with the target.

Terminal deoxynucletidyl transferase (TdT) is overexpressed in some cancer types, where it might compete with pol µ during the mutagenic repair of double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. Here we report the discovery and characterization of pyrrolyl and indolyl diketo acids that specifically target TdT and behave as nucleotide-competitive inhibitors. These compounds show a selective toxicity toward MOLT-4 compared to HeLa cells that correlate well with in vitro selectivity for TdT. The binding site of two of these inhibitors was determined by cocrystallization with TdT, explaining why these compounds are competitive inhibitors of the deoxynucleotide triphosphate (dNTP). In addition, because of the observed dual localization of the phenyl substituent, these studies open the possibility of rationally designing more potent compounds.

A pyrophosphatase activity associated with purified HIV-1 particles.

Treatment of HIV-1 with nucleoside reverse transcription inhibitors leads to the emergence of resistance mutations in the reverse transcriptase (RT) gene. Resistance to 3'-azido-3'-deoxythymidine (AZT) and to a lesser extent to 2'-3'-didehydro-2'-3'-dideoxythymidine is mediated by phosphorolytic excision of the chain terminator. Wild-type RT excises AZT by pyrophosphorolysis, while thymidine-associated resistance mutations in RT (TAMs) favour ATP as the donor substrate. However, in vitro, resistant RT still uses pyrophosphate more efficiently than ATP. We performed in vitro (-) strong-stop DNA synthesis experiments, with wild-type and AZT-resistant HIV-1 RTs, in the presence of physiologically relevant pyrophosphate and/or ATP concentrations and found that in the presence of pyrophosphate, ATP and AZTTP, TAMs do not enhance in vitro (-) strong-stop DNA synthesis. We hypothesized that utilisation of ATP in vivo is driven by intrinsic low pyrophosphate concentrations within the reverse transcription complex, which could be explained by the packaging of a cellular pyrophosphatase. We showed that over-expressed flagged-pyrophosphatase was associated with HIV-1 viral-like particles. In addition, we demonstrated that when HIV-1 particles were purified in order to avoid cellular microvesicle contamination, a pyrophosphatase activity was specifically associated to them. The presence of a pyrophosphatase activity in close proximity to the reverse transcription complex is most likely advantageous to the virus, even in the absence of any drug pressure.

A role of template cleavage in reduced excision of chain-terminating nucleotides by human immunodeficiency virus type 1 reverse transcriptase containing the M184V mutation.

Resistance to nucleoside reverse transcriptase (RT) inhibitors is conferred on human immunodeficiency virus type 1 through thymidine analogue resistance mutations (TAMs) that increase the ability of RT to excise chain-terminating nucleotides after they have been incorporated. The RT mutation M184V is a potent suppressor of TAMs. In RT containing TAMs, the addition of M184V suppressed the excision of 3'-deoxy-3'-azidothymidine monophosphate (AZTMP) to a greater extent on an RNA template than on a DNA template with the same sequence. The catalytically inactive RNase H mutation E478Q abolished this difference. The reduction in excision activity was similar with either ATP or pyrophosphate as the acceptor substrate. Decreased excision of AZTMP was associated with increased cleavage of the RNA template at position -7 relative to the primer terminus, which led to increased primer-template dissociation. Whether M184V was present or not, RT did not initially bind at the -7 cleavage site. Cleavage at the initial site was followed by RT dissociation and rebinding at the -7 cleavage site, and the dissociation and rebinding were enhanced when the M184V mutation was present. In contrast to the effect of M184V, the K65R mutation suppressed the excision activity of RT to the same extent on either an RNA or a DNA template and did not alter the RNase H cleavage pattern. Based on these results, we propose that enhanced RNase H cleavage near the primer terminus plays a role in M184V suppression of AZT resistance, while K65R suppression occurs through a different mechanism.

Substrate mimicry: HIV-1 reverse transcriptase recognizes 6-modified-3'-azido-2',3'-dideoxyguanosine-5'-triphosphates as adenosine analogs.

ß-D-3'-Azido-2',3'-dideoxyguanosine (3'-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3'-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3'-azido-ddG in primary cells. To gain insight into their structure-activity-resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3'-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3'-azido-2,6-diaminopurine >3'-azido-6-chloropurine; 3'-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure-activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1.

Structural basis of HIV-1 resistance to AZT by excision.

Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

Structural basis for the efficient phosphorylation of AZT-MP (3'-azido-3'-deoxythymidine monophosphate) and dGMP by Plasmodium falciparum type I thymidylate kinase.

Plasmodium falciparum is the causative agent of malaria, a disease where new drug targets are required due to increasing resistance to current anti-malarials. TMPK (thymidylate kinase) is a good candidate as it is essential for the synthesis of dTTP, a critical precursor of DNA and has been much studied due to its role in prodrug activation and as a drug target. Type I TMPKs, such as the human enzyme, phosphorylate the substrate AZT (3'-azido-3'-deoxythymidine)-MP (monophosphate) inefficiently compared with type II TMPKs (e.g. Escherichia coli TMPK). In the present paper we report that eukaryotic PfTMPK (P. falciparum TMPK) presents sequence features of a type I enzyme yet the kinetic parameters for AZT-MP phosphorylation are similar to those of the highly efficient E. coli enzyme. Structural information shows that this is explained by a different juxtaposition of the P-loop and the azide of AZT-MP. Subsequent formation of the transition state requires no further movement of the PfTMPK P-loop, with no steric conflicts for the azide moiety, allowing efficient phosphate transfer. Likewise, we present results that confirm the ability of the enzyme to uniquely accept dGMP as a substrate and shed light on the basis for its wider substrate specificity. Information resulting from two ternary complexes (dTMP-ADP and AZT-MP-ADP) and a binary complex with the transition state analogue AP5dT [P1-(5'-adenosyl)-P5-(5'-thymidyl) pentaphosphate] all reveal significant differences with the human enzyme, notably in the lid region and in the P-loop which may be exploited in the rational design of Plasmodium-specific TMPK inhibitors with therapeutic potential.

Investigating the biochemical impact of DNA damage with structure-based probes: abasic sites, photodimers, alkylation adducts, and oxidative lesions.

DNA sustains a wide variety of damage, such as the formation of abasic sites, pyrimidine dimers, alkylation adducts, or oxidative lesions, upon exposure to UV radiation, alkylating agents, or oxidative conditions. Since these forms of damage may be acutely toxic or mutagenic and potentially carcinogenic, it is of interest to gain insight into how their structures impact biochemical processing of DNA, such as synthesis, transcription, and repair. Lesion-specific molecular probes have been used to study polymerase-mediated translesion DNA synthesis of abasic sites and TT dimers, while other probes have been developed for specifically investigating the alkylation adduct O(6)-Bn-G and the oxidative lesion 8-oxo-G. In this review, recent examples of lesion-specific molecular probes are surveyed; their specificities of incorporation opposite target lesions compared to unmodified nucleotides are discussed, and limitations of their applications under physiologically relevant conditions are assessed.

Bisphosphonate inhibitors of ATP-mediated HIV-1 reverse transcriptase catalyzed excision of chain-terminating 3'-azido, 3'-deoxythymidine: a QSAR investigation.

We report the results of an investigation of the inhibition of the ATP-mediated HIV-1 reverse transcriptase catalyzed phosphorolysis in vitro of AZT from AZT-terminated DNA primers by a series of 42 bisphosphonates. The four most active compounds possess neutral, halogen-substituted phenyl or biphenyl sidechains and have IC(50) values < 1 microM in excision inhibition assays. Use of two comparative molecular similarity analysis methods to analyze these inhibition results yielded a classification model with an overall accuracy of 94%, and a regression model having good accord with experiment (q(2)=0.63, r(2)=0.91), with the experimental activities being predicted within, on average, a factor of 2. The most active species had little or no toxicity against three human cell lines (IC(50)(avg) > 200 microM). These results are of general interest since they suggest that it may be possible to develop potent bisphosphonate-based AZT-excision inhibitors with little cellular toxicity, opening up a new route to restoring AZT sensitivity in otherwise resistant HIV-1 strains.

Novel FRET-based assay to detect reverse transcriptase activity using modified dUTP analogues.

We have developed a novel continuous assay to measure reverse transcriptase (RT) polymerase activity. The assay uses fluorescence energy transfer measurements to detect the incorporation of complementary pairs of fluorescently labeled deoxyuridine into cDNA product. The fluorescently labeled dUTP substrates were prepared using commercially available reagents with a simple coupling reaction. The fluorescent dye pairs have significant spectral overlap which allows FRET interaction between dyes incorporated into the cDNA. Using a polyA/oligo dT primer/template, the assay can readily detect DNA polymerase activity from any viral reverse transcriptase enzyme. The reaction proceeds linearly over time, and the rate is proportional to the enzyme concentration. We used the assay to compare the thermostability of a number of wild-type and mutant viral RT enzymes. Our results indicate that the wild-type AMV (avian myeloblastosis virus) enzyme is slightly more stable at 43 degrees C than the HIV-1 (human immunodeficiency virus) or MMLV (Moloney murine leukemia virus) enzymes. The thermostability of the RT enzyme was dramatically increased by the presence of primer/template with the enzyme. We also used the assay to study the effects of inhibitors on HIV-1 RT polymerase activity. This assay may be highly useful for the identification and characterization of potent RT inhibitors which could be candidates for development as therapeutic antiviral agents.

AZT resistance of simian foamy virus reverse transcriptase is based on the excision of AZTMP in the presence of ATP.

Azidothymidine (AZT, zidovudine) is one of the few nucleoside inhibitors known to inhibit foamy virus replication. We have shown previously that up to four mutations in the reverse transcriptase gene of simian foamy virus from macaque (SFVmac) are necessary to confer high resistance against AZT. To characterize the mechanism of AZT resistance we expressed two recombinant reverse transcriptases of highly AZT-resistant SFVmac in Escherichia coli harboring three (K211I, S345T, E350K) or four mutations (K211I, I224T, S345T, E350K) in the reverse transcriptase gene. Our analyses show that the polymerization activity of these mutants is impaired. In contrast to the AZT-resistant reverse transcriptase of HIV-1, the AZT resistant enzymes of SFVmac reveal differences in their kinetic properties. The SFVmac enzymes exhibit lower specific activities on poly(rA)/oligo(dT) and higher K(M)-values for polymerization but no change in K(D)-values for DNA/DNA or RNA/DNA substrates. The AZT resistance of the mutant enzymes is based on the excision of the incorporated inhibitor in the presence of ATP. The additional amino acid change of the quadruple mutant appears to be important for regaining polymerization efficiency.

Tobacco proliferating cell nuclear antigen binds directly and stimulates both activity and processivity of ddNTP-sensitive mungbean DNA polymerase.

PCNA is well known as a component of DNA replication system and plays important roles in multiple cellular pathways in addition to replication and repair. In this work we have demonstrated the physical and functional interaction between tobacco PCNA and mungbean ddNTP-sensitive DNA polymerase which shares many physicochemical properties with family X-DNA polymerases except with the moderately processive mode of nucleotide incorporation. We have shown here that recombinant PCNA binds directly to mungbean DNA polymerase as revealed in affinity chromatography, pull-down and co-immunoprecipitation approaches. In vitro DNA polymerase activity assay and processivity analyses indicated recombinant PCNA specifically stimulates both activity and processivity of mungbean DNA polymerase. These observations lead to interesting speculation about the functional significance of the ddNTP-sensitive enzyme in replication event in higher plants since the enzyme has been shown to be active and expressed at an elevated level during the endoreduplication stages in developing mungbean seeds.

Intracellular metabolism and persistence of the anti-human immunodeficiency virus activity of 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine, a novel thymidine analog.

The therapeutic benefits of current antiretroviral therapy are limited by the evolution of drug-resistant virus and long-term toxicity. Novel antiretroviral compounds with activity against drug-resistant viruses are needed. 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a novel thymidine analog, has potent anti-human immunodeficiency virus (HIV) activity, maintains considerable activity against multidrug-resistant HIV strains, and is less inhibitory to mitochondrial DNA synthesis in cell culture than its progenitor stavudine (D4T). We investigated the intracellular metabolism and anti-HIV activity of 4'-Ed4T. The profile of 4'-Ed4T metabolites was qualitatively similar to that for zidovudine (AZT), with the monophosphate metabolite as the major metabolite, in contrast to that for D4T, with relatively poor formation of total metabolites. The first phosphorylation step for 4'-Ed4T in cells was more efficient than that for D4T but less than that for AZT. The amount of 4'-Ed4T triphosphate (4'-Ed4TTP) was higher than that of AZTTP at 24 h in culture. There was a dose-dependent accumulation of 4'-Ed4T diphosphate and 4'-Ed4TTP on up-regulation of thymidylate kinase and 3-phosphoglycerate kinase expression in Tet-On RKO cells, respectively. The anti-HIV activity of 4'-Ed4T in cells persisted even after 48 h of drug removal from culture in comparison with AZT, D4T, and nevirapine (NVP). The order of increasing persistence of anti-HIV activity of these compounds after drug removal was 4'-Ed4T > D4T > AZT > NVP. In conclusion, with the persistence of 4'-Ed4TTP and persistent anti-HIV activity in cells, we anticipate less frequent dosing and fewer patient compliance issues than for D4T. 4'-Ed4T is a promising antiviral candidate for HIV type 1 chemotherapy.

Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides.

This unit presents synthetic procedures for the preparation of 5'-amino-2',5'-dideoxy analogs of adenosine, cytidine, guanosine, and thymidine, as well as corresponding 5'-N-triphosphate nucleotides, using commercially available reagents. The modified nucleosides are prepared in high yields from naturally occurring 2'-deoxynucleosides using robust chemical reactions including tosylation, azide exchange, and the Staudinger reaction. Efficient conversion of these 5'-amino nucleosides to corresponding 5'-N-triphosphate nucleotides is achieved through a one-step reaction with trimetaphosphate in Tris-buffered aqueous solution. The 5'-amino modification renders these nucleoside and nucleotide analogs markedly increased reactivity, which is useful for a variety of biochemical, pharmaceutical, and genomic applications. Also included in this unit are protocols for polymerase incorporation of the 5'-amino nucleotides, either partially or completely replacing their naturally occurring counterparts, and subsequent sequence-specific cleavage at the modified nucleotides under mildly acidic conditions.