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MutT homologue 1 (MTH1) removes oxidized nucleotides from the nucleotide pool and thereby prevents their incorporation into the genome and thereby reduces genotoxicity. We previously reported that MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis suggesting that MTH1 may also sanitize the nucleotide pool from other methylated nucleotides. We here show that MTH1 efficiently catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and further report that N6-methylation of dATP drastically increases the MTH1 activity. We also observed MTH1 activity with N6-methyl-ATP, albeit at a lower level. We show that N6-methyl-dATP is incorporated into DNA in vivo , as indicated by increased N6-methyl-dA DNA levels in embryos developed from MTH1 knock-out zebrafish eggs microinjected with N6-methyl-dATP compared with noninjected embryos. N6-methyl-dATP activity is present in MTH1 homologues from distantly related vertebrates, suggesting evolutionary conservation and indicating that this activity is important. Of note, N6-methyl-dATP activity is unique to MTH1 among related NUDIX hydrolases. Moreover, we present the structure of N6-methyl-dAMP-bound human MTH1, revealing that the N6-methyl group is accommodated within a hydrophobic active-site subpocket explaining why N6-methyl-dATP is a good MTH1 substrate. N6-methylation of DNA and RNA has been reported to have epigenetic roles and to affect mRNA metabolism. We propose that MTH1 acts in concert with adenosine deaminase-like protein isoform 1 (ADAL1) to prevent incorporation of N6-methyl-(d)ATP into DNA and RNA. This would hinder potential dysregulation of epigenetic control and RNA metabolism via conversion of N6-methyl-(d)ATP to N6-methyl-(d)AMP, followed by ADAL1-catalyzed deamination producing (d)IMP that can enter the nucleotide salvage pathway., (© 2020 Scaletti et al.)

Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain- and HD domain-containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase-mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for ∼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages., (© 2020 Oo et al.)

SAMHD1 is a dNTP hydrolase, whose activity is required for maintaining low dNTP concentrations in non-cycling T cells, dendritic cells, and macrophages. SAMHD1-dependent dNTP depletion is thought to impair retroviral replication in these cells, but the relationship between the dNTPase activity and retroviral restriction is not fully understood. In this study, we investigate allosteric activation of SAMHD1 by deoxynucleotide-dependent tetramerization and measure how the lifetime of the enzymatically active tetramer is affected by different dNTP ligands bound in the allosteric site. The EC 50 dNTP values for SAMHD1 activation by dNTPs are in the 2-20 μm range, and the half-life of the assembled tetramer after deoxynucleotide depletion varies from minutes to hours depending on what dNTP is bound in the A2 allosteric site. Comparison of the wild-type SAMHD1 and the T592D mutant reveals that the phosphomimetic mutation affects the rates of tetramer dissociation, but has no effect on the equilibrium of allosteric activation by deoxynucleotides. Collectively, our data suggest that deoxynucleotide-dependent tetramerization contributes to regulation of deoxynucleotide levels in cycling cells, whereas in non-cycling cells restrictive to retroviral replication, SAMHD1 activation is likely to be achieved through a distinct mechanism., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) plays a critical role in inhibiting HIV infection, curtailing the pool of dNTPs available for reverse transcription of the viral genome. Recent structural data suggested a compelling mechanism for the regulation of SAMHD1 enzymatic activity and revealed dGTP-induced association of two inactive dimers into an active tetrameric enzyme. Here, we present the crystal structures of SAMHD1 catalytic core (residues 113-626) tetramers, complexed with mixtures of nucleotides, including dGTP/dATP, dGTP/dCTP, dGTP/dTTP, and dGTP/dUTP. The combined structural and biochemical data provide insight into dNTP promiscuity at the secondary allosteric site and how enzymatic activity is modulated. In addition, we present biochemical analyses of GTP-induced SAMHD1 full-length tetramerization and the structure of SAMHD1 catalytic core tetramer in complex with GTP/dATP, revealing the structural basis of GTP-mediated SAMHD1 activation. Altogether, the data presented here advance our understanding of SAMHD1 function during cellular homeostasis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase ι (pol ι), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol ι to incorporate NTPs during DNA synthesis. pol ι incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A "steric gate" pol ι mutant is considerably more active in the presence of Mn(2+) compared with Mg(2+) and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol ι is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol ι incorporates NTPs in a template-specific manner, certain DNA sequences may be "at risk" for elevated mutagenesis during pol ι-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol ι becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations.

HIV-1 proviral DNA integration into host chromosomal DNA is only partially completed by the viral integrase, leaving two single-stranded DNA gaps with 5'-end mismatched viral DNA flaps. It has been inferred that these gaps are repaired by the cellular DNA repair machinery. Here, we investigated the efficiency of gap repair at integration sites in different HIV-1 target cell types. First, we found that the general gap repair machinery in macrophages was attenuated compared with that in dividing CD4(+) T cells. In fact, the repair in macrophages was heavily reliant upon host DNA polymerase β (Pol β). Second, we tested whether the poor dNTP availability found in macrophages is responsible for the delayed HIV-1 proviral DNA integration in this cell type because the Km value of Pol β is much higher than the dNTP concentrations found in macrophages. Indeed, with the use of a modified quantitative AluI PCR assay, we demonstrated that the elevation of cellular dNTP concentrations accelerated DNA gap repair in macrophages at HIV-1 proviral DNA integration sites. Finally, we found that human monocytes, which are resistant to HIV-1 infection, exhibited severely restricted gap repair capacity due not only to the very low levels of dNTPs detected but also to the significantly reduced expression of Pol β. Taken together, these results suggest that the low dNTP concentrations found in macrophages and monocytes can restrict the repair steps necessary for HIV-1 integration.

HIV-1 reverse transcriptase (RT) frequently incorporates ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, particularly under the limited dNTP conditions found in macrophages. We investigated the mechanistic impacts of an rNMP embedded in DNA templates on HIV-1 RT-mediated DNA synthesis. We observed that the template-embedded rNMP induced pausing of RT and delayed DNA synthesis kinetics at low macrophage dNTP concentrations but not at high T cell dNTP concentrations. Although the binding affinity of RT to the rNMP-containing template-primer was not altered, the dNTP incorporation kinetics of RT were significantly reduced at one nucleotide upstream and downstream of the rNMP site, leading to pause sites. Finally, HIV-1 RT becomes more error-prone at rNMP sites with an elevated mismatch extension capability but not enhanced misinsertion capability. Together these data suggest that rNMPs embedded in DNA templates may influence reverse transcription kinetics and impact viral mutagenesis in macrophages.

Deoxyribonucleoside triphosphate pools in mammalian mitochondria are highly asymmetric, and this asymmetry probably contributes to the elevated mutation rate for the mitochondrial genome as compared with the nuclear genome. To understand this asymmetry, we must identify pathways for synthesis and accumulation of dNTPs within mitochondria. We have identified ribonucleotide reductase activity specifically associated with mammalian tissue mitochondria. Examination of immunoprecipitated proteins by mass spectrometry revealed R1, the large ribonucleotide reductase subunit, in purified mitochondria. Significant enzymatic and immunological activity was seen in rat liver mitochondrial nucleoids, isolated as described by Wang and Bogenhagen (Wang, Y., and Bogenhagen, D. F. (2006) J. Biol. Chem. 281, 25791-25802). Moreover, incubation of respiring rat liver mitochondria with [(14)C]cytidine diphosphate leads to accumulation of radiolabeled deoxycytidine and thymidine nucleotides within the mitochondria. Comparable results were seen with [(14)C]guanosine diphosphate. Ribonucleotide reduction within the mitochondrion, as well as outside the organelle, needs to be considered as a possibly significant contributor to mitochondrial dNTP pools.

A large number of nucleoside analogues and 2'-deoxynucleoside triphosphates (dNTP) have been synthesized to interfere with DNA metabolism. However, in vivo the concentration and phosphorylation of these analogues are key limiting factors. In this context, we designed enzymes to switch nucleobases attached to a deoxyribose monophosphate. Active chimeras were made from two distantly related enzymes: a nucleoside deoxyribosyltransferase from lactobacilli and a 5'-monophosphate-2'-deoxyribonucleoside hydrolase from rat. Then their unprecedented activity was further extended to deoxyribose triphosphate, and in vitro biosyntheses could be successfully performed with several base analogues. These new enzymes provide new tools to synthesize dNTP analogues and to deliver them into cells.

Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ~12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) M obtained with dTTP for dTMP kinase is ~3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) M), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.

Several physiologically relevant cations including Ca(2+), Mn(2+), and Zn(2+) have been shown to inhibit HIV reverse transcriptase (RT), presumably by competitively displacing one or more Mg(2+) ions bound to RT. We analyzed the effects of Zn(2+) on reverse transcription and compared them to Ca(2+) and Mn(2+). Using nucleotide extension efficiency as a readout, Zn(2+) showed significant inhibition of reactions with 2 mM Mg(2+), even when present at only ∼5 μM. Mn(2+) and Ca(2+) were also inhibitory but at higher concentrations. Both Mn(2+) and Zn(2+) (but not Ca(2+)) supported RT incorporation in the absence of Mg(2+) with Mn(2+) being much more efficient. The maximum extension rates with Zn(2+), Mn(2+), and Mg(2+) were ∼0.1, 1, and 3.5 nucleotides per second, respectively. Zinc supported optimal RNase H activity at ∼25 μM, similar to the optimal for nucleotide addition in the presence of low dNTP concentrations. Surprisingly, processivity (average number of nucleotides incorporated in a single binding event with enzyme) during reverse transcription was comparable with Zn(2+) and Mg(2+), and single RT molecules were able to continue extension in the presence of Zn(2+) for several hours on the same template. Consistent with this result, the half-life for RT-Zn(2+)-(primer-template) complexes was 220 ± 60 min and only 1.7 ± 1 min with Mg(2+), indicating ∼130-fold more stable binding with Zn(2+). Essentially, the presence of Zn(2+) promotes the formation of a highly stable slowly progressing RT-(primer-template) complex.

During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2'-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3-14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3'-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed.

Ribonucleotide reduction provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and DNA repair. In cycling mammalian cells the reaction is catalyzed by two proteins, R1 and R2. A third protein, p53R2, with the same function as R2, occurs in minute amounts. In quiescent cells, p53R2 replaces the absent R2. In humans, genetic inactivation of p53R2 causes early death with mtDNA depletion, especially in muscle. We found that cycling fibroblasts from a patient with a lethal mutation in p53R2 contained a normal amount of mtDNA and showed normal growth, ribonucleotide reduction, and deoxynucleoside triphosphate (dNTP) pools. However, when made quiescent by prolonged serum starvation the mutant cells strongly down-regulated ribonucleotide reduction, decreased their dCTP and dGTP pools, and virtually abolished the catabolism of dCTP in substrate cycles. mtDNA was not affected. Also, nuclear DNA synthesis and the cell cycle-regulated enzymes R2 and thymidine kinase 1 decreased strongly, but the mutant cell populations retained unexpectedly larger amounts of the two enzymes than the controls. This difference was probably due to their slightly larger fraction of S phase cells and therefore not induced by the absence of p53R2 activity. We conclude that loss of p53R2 affects ribonucleotide reduction only in resting cells and leads to a decrease of dNTP catabolism by substrate cycles that counterweigh the loss of anabolic activity. We speculate that this compensatory mechanism suffices to maintain mtDNA in fibroblasts but not in muscle cells with a larger content of mtDNA necessary for their high energy requirements.

The 2',3'-dideoxy-3'-thiacytidine drug-resistant M184I HIV-1 reverse transcriptase (RT) has been shown to synthesize DNA with decreased processivity compared with the wild-type RT. M184A displays an even more severe processivity defect. However, the basis of this decreased processivity has been unclear, and both primer-template binding and dNTP interaction defects have been proposed to account for it. In this study, we show that the altered properties of the M184I and M184A RT mutants that we have measured, including decreased processivity, a slower rate of primer extension, and increased strand transfer activity, can all be explained by a defect in dNTP utilization. These alterations are observed only at low dNTP concentration and vanish as the dNTP concentration is raised. The mutant RTs exhibit a normal dissociation rate from a DNA primer-RNA template while paused during synthesis. Slower than normal synthesis at physiological dNTP concentration, coupled with normal dissociation from the primer-template, results in the lowered processivity. The mutant RTs exhibit normal DNA 3'-end-directed and RNA 5'-end-directed ribonuclease H activity. The reduced rate of DNA synthesis causes an increase in the ratio of ribonuclease H to polymerase activity thereby promoting increased strand transfer. These latter results are consistent with an observed higher rate of recombination by HIV-1 strains with Met-184 mutations.

We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (-)-2',3'-deoxy-3'-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a approximately 10- to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the k(pol) values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid beta-branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.

We have characterized, by transient-state kinetic methods, the polymerase and exonuclease activities of the human mitochondrial DNA polymerase (pol gamma) during reverse transcription, employing a synthetic oligonucleotide consisting of a DNA primer and an RNA template. In comparison with the kinetic parameters observed with a DNA template, the rate of correct deoxynucleotide incorporation was reduced 25-fold (5.5+/-0.2 s(-1)), whereas the dissociation constant (Kd) for nucleotide binding was increased 4-fold (12+/-1 microm). In addition, discrimination against mismatches was reduced approximately 20-fold to only 15,000 on average. The proofreading exonuclease favored the removal of an incorrect nucleotide (0.0021+/-0.0002 s(-1) for correct versus 0.034+/-0.004 s(-1) for incorrect), and the partitioning between incorporation beyond a mismatch (5.5x10(-5)+/-0.4x10(-5) s(-1)), and exonuclease removal of that mismatch favors removal of the mismatch. These data suggest that the "reverse transcriptase activity" of mitochondrial polymerase could be physiologically relevant. However, the enzyme stalls and is unable to efficiently incorporate beyond a single nucleotide with an RNA template. Additionally, we present a refined method for calculating net discrimination, which more accurately describes the contributions of correct and incorrect incorporation. The biological and biotechnological significance of these results are discussed.

The SWI/SNF chromatin remodeling complex plays a critical role in the coordination of gene expression with physiological stimuli. The synthetic enzymes ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase are coordinately regulated to ensure appropriate deoxyribonucleotide triphosphate levels. Particularly, these enzymes are actively repressed as cells exit the cell cycle through the action of E2F transcription factors and the retinoblastoma tumor suppressor/p107/p130 family of pocket proteins. This process is found to be highly dependent on SWI/SNF activity as cells deficient in BRG-1 and Brm subunits fail to repress these genes with activation of pocket proteins, and this deficit in repression can be complemented, via the ectopic expression of BRG-1. The failure to repress transcription does not involve a blockade in the association of E2F or pocket proteins p107 and p130 with promoter elements. Rather, the deficit in repression is due to a failure to mediate histone deacetylation of ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase promoters in the absence of SWI/SNF activity. The basis for this is found to be a failure to recruit mSin3B and histone deacetylase proteins to promoters. Thus, the coordinate repression of deoxyribonucleotide triphosphate metabolic enzymes is dependent on the action of SWI/SNF in facilitating the assembly of repressor complexes at the promoter.

Human fibroblasts in culture obtain deoxynucleotides by de novo ribonucleotide reduction or by salvage of deoxynucleosides. In cycling cells the de novo pathway dominates, but in quiescent cells the salvage pathway becomes important. Two forms of active mammalian ribonucleotide reductases are known. Each form contains the catalytic R1 protein, but the two differ with respect to the second protein (R2 or p53R2). R2 is cell cycle-regulated, degraded during mitosis, and absent from quiescent cells. The recently discovered p53-inducible p53R2 was proposed to be linked to DNA repair processes. The protein is not cell cycle-regulated and can provide deoxynucleotides to quiescent mouse fibroblasts. Here we investigate the in situ activities of the R1-p53R2 complex and two other enzymes of the de novo pathway, dCMP deaminase and thymidylate synthase, in confluent quiescent serum-starved human fibroblasts in experiments with [5-(3)H]cytidine, [6-(3)H]deoxycytidine, and [C(3)H(3)]thymidine. These cells had increased their content of p53R2 2-fold and lacked R2. From isotope incorporation, we conclude that they have a complete de novo pathway for deoxynucleotide synthesis, including thymidylate synthesis. During quiescence, incorporation of deoxynucleotides into DNA was very low. Deoxynucleotides were instead degraded to deoxynucleosides and exported into the medium as deoxycytidine, deoxyuridine, and thymidine. The rate of export was surprisingly high, 25% of that in cycling cells. Total ribonucleotide reduction in quiescent cells amounted to only 2-3% of cycling cells. We suggest that in quiescent cells an important function of p53R2 is to provide deoxynucleotides for mitochondrial DNA replication.

Previous work has shown that Sulfolobus solfataricus DNA polymerase Dpo4-catalyzed bypass of O(6)-methylguanine (O(6)-MeG) proceeds largely in an accurate but inefficient manner with a "wobble" base pairing between C and O(6)-MeG (Eoff, R. L., Irimia, A., Egli, M., and Guengerich, F. P. (2007) J. Biol. Chem. 282, 1456-1467). We considered here the bulky lesion O(6)-benzylguanine (O(6)-BzG) in DNA and catalysis by Dpo4. Mass spectrometry analysis of polymerization products revealed that the enzyme bypasses and extends across from O(6)-BzG, with C the major product ( approximately 70%) and some T and A ( approximately 15% each) incorporated opposite the lesion. Steady-state kinetic parameters indicated that Dpo4 was 7-, 5-, and 27-fold more efficient at C incorporation opposite O(6)-BzG than T, A, or G, respectively. In transient state kinetic analysis, the catalytic efficiency was decreased 62-fold for C incorporation opposite O(6)-BzG relative to unmodified DNA. Crystal structures reveal wobble pairing between C and O(6)-BzG. Pseudo-"Watson-Crick" pairing was observed between T and O(6)-BzG. Two other structures illustrate a possible mechanism for the accommodation of a +1 frameshift in the Dpo4 active site. The overall effect of O(6)-BzG is to decrease the efficiency of bypass by roughly an order of magnitude in every case except correct bypass, where the effect is not as pronounced. By comparison, Dpo4 is more accurate but no more efficient than model replicative polymerases, such as bacteriophage T7(-) DNA polymerase and human immunodeficiency virus-1 reverse transcriptase in the polymerization past O(6)-MeG and O(6)-BzG.

The RNase H activity of human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) cleaves the viral genome concomitant with minus strand synthesis. We previously analyzed RT-mediated pausing and RNase H cleavage on a hairpin-containing RNA template system and reported that RT generated 3' end-directed primary and secondary cuts while paused at the base of the hairpin during synthesis. Here, we report that all of the prominent cleavage products observed during primer extension on this template correlated with pause induced cuts. Products that persisted throughout the reaction corresponded to secondary cuts, about eight nucleotides in from the DNA primer terminus. This distance allows little overlap of intact template with the primer terminus. We considered whether secondary cuts could inactivate further synthesis by promoting dissociation of the primer from the template. As anticipated, 3' end-directed secondary cuts decreased primer extendibility. This provides a plausible mechanism to explain the persistence of secondary cut products in our hairpin template system. Improving the efficiency of synthesis by increasing the concentration of dNTPs or addition of nucleocapsid protein (NC) reduced pausing and the generation of pause related secondary cuts on this template. Further studies reveal that 3' end-directed primary and secondary cleavages were also generated when synthesis was stalled by the presence of 3'-azido-3'-deoxythymidine at the primer terminus, possibly contributing to 3'-azido-3'-deoxythymidine inhibition. Considered together, the data reveal a role for NC and other factors that enhance DNA synthesis in the prevention of RNase H cleavages that could be detrimental to viral replication.

We have recently demonstrated that HIV-1 RT mutants characterized by low dNTP binding affinity display significantly reduced dNTP incorporation kinetics in comparison to wild-type RT. This defect is particularly emphasized at low dNTP concentrations where WT RT remains capable of efficient synthesis. Kinetic interference in DNA synthesis can induce RT pausing and slow down the synthesis rate. RT stalling and slow synthesis rate can enhance RNA template cleavage by RT-RNase H, facilitating transfer of the primer to a homologous template. We therefore hypothesized that reduced dNTP binding RT mutants can promote template switching during minus strand synthesis more efficiently than WT HIV-1 RT at low dNTP concentrations. To test this hypothesis, we employed two dNTP binding HIV-1 RT mutants, Q151N and V148I. Indeed, as the dNTP concentration was decreased, the template switching frequency progressively increased for both WT and mutant RTs. However, as predicted, the RT mutants promoted more transfers compared with WT RT. The WT and mutant RTs were similar in their intrinsic RNase H activity, supporting that the elevated template switching efficiency of the mutants was not the result of the mutations enhancing RNase H activity. Rather, kinetic interference leading to stalled DNA synthesis likely enhanced transfers. These results suggest that the RT-dNTP substrate interaction mechanistically influences strand transfer and recombination of HIV-1 RT.

Thioredoxin was initially identified by its ability to serve as an electron donor for ribonucleotide reductase in vitro. Whether it serves a similar function in vivo is unclear. In Saccharomyces cerevisiae, it was previously shown that Deltatrx1 Deltatrx2 mutants lacking the two genes for cytosolic thioredoxin have a slower growth rate because of a longer S phase, but the basis for S phase elongation was not identified. The hypothesis that S phase protraction was due to inefficient dNTP synthesis was investigated by measuring dNTP levels in asynchronous and synchronized wild-type and Deltatrx1 Deltatrx2 yeast. In contrast to wild-type cells, Deltatrx1 Deltatrx2 cells were unable to accumulate or maintain high levels of dNTPs when alpha-factor- or cdc15-arrested cells were allowed to reenter the cell cycle. At 80 min after release, when the fraction of cells in S phase was maximal, the dNTP pools in Deltatrx1 Deltatrx2 cells were 60% that of wild-type cells. The data suggest that, in the absence of thioredoxin, cells cannot support the high rate of dNTP synthesis required for efficient DNA synthesis during S phase. The results constitute in vivo evidence for thioredoxin being a physiologically relevant electron donor for ribonucleotide reductase during DNA precursor synthesis.

We have measured the stabilities of the bonds that are critical for determining the half-life of ribonucleotides and the beta-glycosidic and 3'- and 5'-phosphoester bonds. Stabilities were measured under a wide range of temperatures and water/formamide ratios. The stability of phosphodiester bonds in oligoribonucleotides was determined in the same environments. The comparison of bond stabilities in the monomer versus the polymer forms of the ribo compounds revealed that physico-chemical conditions exist in which polymerization is thermodynamically favored. These conditions were compared with those determining a similar behavior for 2'-deoxyribonucleosides, deoxyribonucleotides, and deoxyribooligonucleotides and were shown to profoundly differ. The implications of these facts on the origin of informational polymers are discussed.

Ribonucleotide reductase (RNR) is an essential enzyme that provides the cell with a balanced supply of deoxyribonucleoside triphosphates for DNA replication and repair. Mutations that affect the regulation of RNR in yeast and mammalian cells can lead to genetic abnormalities and cell death. We have expressed and purified the components of the RNR system in fission yeast, the large subunit Cdc22p, the small subunit Suc22p, and the replication inhibitor Spd1p. It was proposed (Liu, C., Powell, K. A., Mundt, K., Wu, L., Carr, A. M., and Caspari, T. (2003) Genes Dev. 17, 1130-1140) that Spd1 is an RNR inhibitor, acting by anchoring the Suc22p inside the nucleus during G1 phase. Using in vitro assays with highly purified proteins we have demonstrated that Spd1 indeed is a very efficient inhibitor of fission yeast RNR, but acting on Cdc22p. Furthermore, biosensor technique showed that Spd1p binds to the Cdc22p with a KD of 2.4 microM, whereas the affinity to Suc22p is negligible. Therefore, Spd1p inhibits fission yeast RNR activity by interacting with the Cdc22p. Similar to the situation in budding yeast, logarithmically growing fission yeast increases the dNTP pools 2-fold after 3 h of incubation in the UV mimetic 4-nitroquinoline-N-oxide. This increase is smaller than the increase observed in budding yeast but of the same order as the dNTP pool increase when synchronous Schizosaccharomyces pombe cdc10 cells are going from G1 to S-phase.

To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme superfamilies: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0-8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn(2+) > Co(2+) > Ni(2+) > Mg(2+); YfbR: Co(2+) > Mn(2+) > Cu(2+); YjjG: Mg(2+) > Mn(2+) > Co(2+)). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo- and deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates with highest affinity to 3'-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P(20-25)). YfbR was strictly specific to deoxyribonucleoside 5'-monophosphates, whereas YjjG showed narrow specificity to 5'-dTMP, 5'-dUMP, and 5'-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.

Retroviruses utilize cellular dNTPs to perform proviral DNA synthesis in infected host cells. Unlike oncoretroviruses, which replicate in dividing cells, lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus, are capable of efficiently replicating in non-dividing cells (terminally differentiated macrophages) as well as dividing cells (i.e. activated CD4+ T cells). In general, non-dividing cells are likely to have low cellular dNTP content compared with dividing cells. Here, by employing a novel assay for cellular dNTP content, we determined the dNTP concentrations in two HIV-1 target cells, macrophages and activated CD4+ T cells. We found that human macrophages contained 130-250-fold lower dNTP concentrations than activated human CD4+ T cells. Biochemical analysis revealed that, unlike oncoretroviral reverse transcriptases (RTs), lentiviral RTs efficiently synthesize DNA even in the presence of the low dNTP concentrations equivalent to those found in macrophages. In keeping with this observation, HIV-1 vectors containing mutant HIV-1 RTs, which kinetically mimic oncoretroviral RTs, failed to transduce human macrophages despite retaining normal infectivity for activated CD4+ T cells and other dividing cells. These results suggest that the ability of HIV-1 to infect macrophages, which is essential to establishing the early pathogenesis of HIV-1 infection, depends, at least in part, on enzymatic adaptation of HIV-1 RT to efficiently catalyze DNA synthesis in limited cellular dNTP substrate environments.

Three models describing frameshift mutations are "classical" Streisinger slippage, proposed for repetitive DNA, and "misincorporatation misalignment" and "dNTP-stabilized misalignment," proposed for non-repetitive DNA. We distinguish between models using pre-steady state fluorescence kinetics to visualize transiently misaligned DNA intermediates and nucleotide incorporation products formed by DNA polymerases adept at making small frameshift mutations in vivo. Human polymerase (pol) mu catalyzes Streisinger slippage exclusively in repetitive DNA, requiring as little as a dinucleotide repeat. Escherichia coli pol IV uses dNTP-stabilized misalignment in identical repetitive DNA sequences, revealing that pol mu and pol IV use different mechanisms in repetitive DNA to achieve the same mutational end point. In non-repeat sequences, pol mu switches to dNTP-stabilized misalignment. pol beta generates -1 frameshifts in "long" repeats and base substitutions in "short" repeats. Thus, two polymerases can use two different frameshift mechanisms on identical sequences, whereas one polymerase can alternate between frameshift mechanisms to process different sequences.

Replication forks often stall at undamaged or damaged template sites in Escherichia coli. Subsequent resumption of DNA synthesis occurs by replacing DNA polymerase III, which is bound to DNA by the beta-sliding clamp, with one of three damage-induced DNA polymerases II, IV, or V. The principal role of the beta clamp is to tether the normally weakly bound polmerases to DNA thereby increasing their processivities. DNA polymerase IV binds dNTP substrates with about 10-fold lower affinity compared with the other E. coli polymerases, which if left unchecked could hinder its ability to synthesize DNA in vivo. Here we report a new property for the beta clamp, which when bound to DNA polymerase IV results in a large increase in dNTP binding affinity that concomitantly increases the efficiency of nucleotide incorporation at normal and transiently slipped mispaired primer/template ends. Primer-template DNA slippage resulting in single nucleotide deletions is a biological hallmark of DNA polymerase IV infidelity responsible for enhancing cell fitness in response to stress. We show that the increased DNA polymerase IV-dNTP binding affinity is an intrinsic property of the DNA polymerase IV-beta clamp interaction and not an indirect consequence of an increased binding of DNA polymerase IV to DNA.

Ribonucleotide reductase catalyzes the production of deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates for DNA synthesis. Mammalian ribonucleotide reductase (RNR) is a tetramer consisting of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. We have demonstrated that DNA damage by doxorubicin and cisplatin caused a steady elevation of the R2 protein in p53(-/-) HCT-116 human colon carcinoma cells but induced degradation of the protein in p53(+/+) cells. To evaluate the involvement of R2 in response to DNA damage, p53(-/-) HCT-116 cells were stably transfected with an expression vector transcribing short hairpin/short interference RNA directed against R2 mRNA. Stably transfected clones exhibited a pronounced reduction of the R2 protein with no change in the cellular growth rate. Furthermore, short interference RNA-mediated reduction of the R2 protein caused a marked increase in sensitivity to the DNA-damaging agent cisplatin as well as to the RNR inhibitors Triapine and hydroxyurea. Ectopic expression of p53R2 partially reversed the cytotoxicity of cisplatin but not that of RNR inhibitors to R2 knockdown cells. The increase in sensitivity to cisplatin and RNR inhibitors was correlated with the suppression of dATP and dGTP levels caused by stable expression of R2-targeted short interference RNA. These results indicated that DNA damage resulted in elevated levels of the R2 protein and dNTPs and, consequently, enhanced the survival of p53(-/-) HCT-116 cells. The findings provide evidence that R2-RNR can be employed to supply dNTPs for the repair of DNA damage in cells with an impaired p53-dependent induction of p53R2.

We quantify cytosolic and mitochondrial deoxyribonucleoside triphosphates (dNTPs) from four established cell lines using a recently described method for the separation of cytosolic and mitochondrial (mt) dNTPs from as little as 10 million cells in culture (Pontarin, G., Gallinaro, L., Ferraro, P., Reichard, P., and Bianchi, V. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 12159-12164). In cycling cells the concentrations of the phosphates of thymidine, deoxycytidine, and deoxyadenosine (combining mono-, di-, and triphosphates in each case) did not differ significantly between mitochondria and cytosol, whereas deoxyguanosine phosphates were concentrated to mitochondria. We study the source and regulation of the mt dTTP pool as an example of mt dNTPs. We suggest two pathways as sources for mt dTTP: (i) import from the cytosol of thymidine diphosphate by a deoxynucleotide transporter, predominantly in cells involved in DNA replication with an active synthesis of deoxynucleotides and (ii) import of thymidine followed by phosphorylation by the mt thymidine kinase, predominantly in resting cells. Here we demonstrate that the second pathway is regulated by a mt 5'-deoxyribonucleotidase (mdN). We modify the in situ activity of mdN and measure the transfer of radioactivity from [(3)H]thymidine to mt thymidine phosphates. In cycling cells lacking the cytosolic thymidine kinase, a 30-fold overproduction of mdN decreases the specific radioactivity of mt dTTP to 25%, and an 80% decrease of mdN by RNA interference increases the specific radioactivity 2-fold. These results suggest that mdN modulates the synthesis of mt dTTP by counteracting in a substrate cycle the phosphorylation of thymidine by the mt thymidine kinase.

The relationship between dNTP levels and DNA synthesis was investigated using alpha factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 degrees C before being released to 25 degrees C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G1 levels. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 degrees C were able to replicate when subsequently switched to 37 degrees C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.

The ability of RNA polymerase (RNAP) to adopt multiple conformations is central to transcriptional regulation. In previous work, we demonstrated that RNAP can exist in an unactivated state that catalyzes synthesis slowly and an activated state that catalyzes synthesis rapidly, with the transition from the unactivated to the activated state being induced by the templated NTP binding to an allosteric site on the RNAP. In this work, we investigate the effects of downstream DNA sequences on the kinetics of single nucleotide incorporation. We demonstrate that changing the identity of the DNA base 1 bp downstream (+2) from the site of incorporation (+1) can regulate the catalytic activity of RNAP. Combining these data with sequence and structural analyses and molecular modeling, we identify the streptolydigin-binding region (Escherichia coli beta residues 543-546), which lies across from the downstream DNA, as the putative allosteric NTP binding site. We present a structural model in which the NTP binds to the streptolydigin loop and upon pairing with the +1 DNA base in the unactivated state or the +2 DNA base in the activated state facilitates translocation via a ratchet motion. This model provides an alternative mechanism for pausing as well as a structural explanation not only for our kinetic data but also for data from elongation studies on yeast RNAP II.

Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) that is strictly required for catalytic activity and for genetic complementation of a pol alpha-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol alpha G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol alpha G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol alpha, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol alpha bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus. Furthermore, pol alpha G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6-4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol alpha.

DNA polymerases delta and epsilon (pol delta and epsilon) are the major replicative polymerases and possess 3'-5' proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol epsilon, the 3'-5' exonuclease-deficient pol2-4, a +1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol epsilon was 0.47 x 10(-5) for G:G mismatches, 0.15 x 10(-5) for T:G mismatches, and less than 0.01 x 10(-5) for A:G mismatches. The accuracy for A opposite G was not altered in the exonuclease-deficient pol2-4 pol epsilon; however, G:G and T:G misincorporation rates increased 40- and 73-fold, respectively. The pol2C1089Y pol epsilon mutant also exhibited increased G:G and T:G misincorporation rates, 22- and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3'-5' exonuclease activity of pol2C1089Y pol epsilon is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.

Escherichia coli DNA polymerase IV (pol IV), a member of the error-prone Y family, predominantly generates -1 frameshifts when copying DNA in vitro. T-->G transversions and T-->C transitions are the most frequent base substitutions observed. The in vitro data agree with mutational spectra obtained when pol IV is overexpressed in vivo. Single base deletion and base substitution rates measured in the lacZalpha gene in vitro are, on average, 2 x 10(-4) and 5 x 10(-5), respectively. The range of misincorporation and mismatch extension efficiencies determined kinetically are 10(-3) to 10(-5). The presence of beta sliding clamp and gamma-complex clamp loading proteins strongly enhance pol IV processivity but have no discernible influence on fidelity. By analyzing changes in fluorescence of a 2-aminopurine template base undergoing replication in real time, we show that a "dNTP-stabilized" misalignment mechanism is responsible for making -1 frameshift mutations on undamaged DNA. In this mechanism, a dNTP substrate is paired "correctly" opposite a downstream template base, on a "looped out" template strand instead of mispairing opposite a next available template base. By using the same mechanism, pol IV "skips" past an abasic template lesion to generate a -1 frameshift. A crystal structure depicting dNTP-stabilized misalignment was reported recently for Sulfolubus solfataricus Dpo4, a Y family homolog of Escherichia coli pol IV.

The Dbh polymerase of Sulfolobus solfataricus is a member of the recently described family of low fidelity DNA polymerases involved in bypass of DNA lesions. To investigate the enzymatic properties of Dbh, we characterized the errors made by this polymerase in vitro. Not only is Dbh much less accurate than the "classical" polymerases, but it showed a remarkable tendency to skip over a template pyrimidine positioned immediately 3' to a G residue, generating a single-base deletion. Single-turnover kinetic measurements suggest possible mechanisms. First, Dbh shows a bias in favor of dCTP, such that the rate of incorporation of dCTP opposite a template G is about 10-fold faster than for the other three dNTPs opposite their complementary partners. On a DNA substrate corresponding to a frameshift hotspot, the rate of frameshift insertion of dCTP opposite a template G that is one residue 5' to the expected templating position is approximately equal to the rate of the non-frameshifted C-dGTP insertion. We suspect that the unusual mutational specificity of Dbh (which is shared with other polymerases from the DinB branch of the bypass polymerase family) may be related to the type of DNA lesion(s) that it serves to bypass in vivo.

Terminal deoxynucleotidyl transferase (TdT) catalyzes the condensation of deoxyribonucleotides on 3'-hydroxyl ends of DNA strands in a template-independent manner and adds N-regions to gene segment junctions during V(D)J recombination. Although TdT is able to incorporate a few ribonucleotides in vitro, TdT discrimination between ribo- and deoxyribonucleotides has never been studied. We found that TdT shows only a minor preference for incorporation of deoxyribonucleotides over ribonucleotides on DNA strands. However, incorporation of ribonucleotides alone or in the presence of deoxyribonucleotides generally leads to premature chain termination, reflecting an impeded accommodation of ribo- or mixed ribo/deoxyribonucleic acid substrates by TdT. An essential catalytic aspartate in TdT was identified, which is a first step toward understanding the apparent lack of sugar discrimination by TdT.

Mitochondrial toxicity can result from antiviral nucleotide analog therapy used to control human immunodeficiency virus type 1 infection. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Apparent steady-state K(m) and k(cat) values for insertion of 2',3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZT-TP), 2',3'-dideoxy-CTP (ddCTP), 2',3'-didehydro-TTP (D4T-TP), (-)-2',3'-dideoxy-3'-thiacytidine (3TC-TP), and carbocyclic 2',3'-didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utilized by pol gamma in vitro as efficiently as natural deoxynucleotides, whereas AZT-TP, 3TC-TP, and CBV-TP were only moderate inhibitors of DNA chain elongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV from DNA predicts persistence in vivo following successful incorporation. In contrast, removal of 3'-terminal 3TC residues was 50% as efficient as natural 3' termini. Finally, we observed inhibition of exonuclease activity by concentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termination, persistence of these analogs in DNA and inhibition of exonucleolytic proofreading may also contribute to mitochondrial toxicity.

We compared the allosteric regulation and effector binding properties of wild type R1 protein and R1 protein with a mutation in the "activity site" (D57N) of mouse ribonucleotide reductase. Wild type R1 had two effector-binding sites per polypeptide chain: one site (activity site) for dATP and ATP, with dATP-inhibiting and ATP-stimulating catalytic activity; and a second site (specificity site) for dATP, ATP, dTTP, and dGTP, directing substrate specificity. Binding of dATP to the specificity site had a 20-fold higher affinity than to the activity site. In all these respects, mouse R1 resembles Escherichia coli R1. Results with D57N were complicated by the instability of the protein, but two major changes were apparent. First, enzyme activity was stimulated by both dATP and ATP, suggesting that D57N no longer distinguished between the two nucleotides. Second, the two binding sites for dATP both had the same low affinity for the nucleotide, similar to that of the activity site of wild type R1. Thus the mutation in the activity site had decreased the affinity for dATP at the specificity site, demonstrating the interaction between the two sites.

The structures of open and closed conformations of DNA polymerase beta (pol beta) suggests that the rate of single-nucleotide deletions during synthesis may be modulated by interactions in the DNA minor groove that align the templating base with the incoming dNTP. To test this hypothesis, we measured the single-base deletion error rates of wild-type pol beta and lysine and alanine mutants of Arg(283), whose side chain interacts with the minor groove edge of the templating nucleotide at the active site. The error rates of both mutant enzymes are increased >100-fold relative to wild-type pol beta. Template engineering experiments performed to distinguish among three possible models for deletion formation suggest that most deletions in repetitive sequences by pol beta initiate by strand slippage. However, pol beta also generates deletions by a different mechanism that is strongly enhanced by the substitutions at Arg(283). Analysis of error specificity suggests that this mechanism involves nucleotide misinsertion followed by primer relocation, creating a misaligned intermediate. The structure of pol beta bound to non-gapped DNA also indicates that the templating nucleotide and its downstream neighbor are out of register in the open conformation and this could facilitate misalignment (dNTP or primer terminus) with the next template base.

Xeroderma pigmentosum (XP) patients are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers. The variant form of XP results from mutations in the hRAD30A gene, which encodes the DNA polymerase in humans, hPol(eta). Of the eukaryotic DNA polymerases, only human Pol(eta) and its yeast counterpart have the ability to replicate DNA containing a cis-syn thymine-thymine (T-T) dimer. Here we measure the fidelity of hPol(eta) on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer. Opposite all four nondamaged template bases, hPol(eta) misincorporates nucleotides with a frequency of approximately 10(-2)-10(-3), and importantly, hPol(eta) synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA. The low fidelity of hPol(eta) may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer.

The influenza virus polymerase complex contains a metal ion-dependent endonuclease activity, which generates short capped RNA primer molecules from capped RNA precursors. Previous studies have provided evidence for a two-metal ion mechanism of RNA cleavage, and the data are consistent with a direct interaction of a divalent metal ion with the catalytic water molecule. To refine the model of this active site, we have generated a series of DNA, RNA, and DNA-RNA chimeric molecules to study the role of the 2'-hydroxy groups on nucleic acid substrates of the endonuclease. We could observe specific cleavage of nucleic acid substrates devoid of any 2'-hydroxy groups if they contained a cap structure (m7GpppG) at the 5'-end. The capped DNA endonuclease products were functional as primers for transcription initiation by the influenza virus polymerase. The apparent cleavage rates were about 5 times lower with capped DNA substrates as compared with capped RNA substrates. Cleavage rates with DNA substrates could be increased to RNA levels by substituting the deoxyribosyl moieties immediately 5' and 3' of the cleavage site with ribosyl moieties. Similarly, cleavage rates of RNA substrates could be lowered to DNA levels by exchanging the same two ribosyl groups with deoxyribosyl groups at the cleavage site. These results demonstrate that the 2'-hydroxy groups are not essential for binding and cleavage of nucleic acids by the influenza virus endonuclease, but small differences of the nucleic acid conformation in the endonuclease active site can influence the overall rate of hydrolysis. The observed relative cleavage rates with DNA and RNA substrates argue against a direct interaction of a catalytic metal ion with a 2'-hydroxy group in the endonuclease active site.

Arginine 72 in human immunodeficiency virus type 1 reverse transcriptase (RT), a highly conserved residue among retroviral polymerases and telomerases, forms part of the binding pocket for the nascent base pair. We show here that replacement of Arg(72) by alanine strongly alters fidelity in a highly unusual manner. R72A reverse transcriptase is a frameshift and base substitution antimutator polymerase whose increased fidelity results both from increased nucleotide selectivity and from a decreased ability to extend mismatched primer termini. Thus, Arg(72)-substrate interactions in wild-type human immunodeficiency virus type 1 RT can stabilize incorrect nucleotides allowing misinsertion and promoting extension of mismatched and perhaps misaligned template-primers. In contrast to the higher fidelity at most sites, R72A RT is highly error-prone for misincorporations opposite template T in the sequence context: 5'-CTGG. Surprisingly, this results mostly from a 1200-fold increase in the apparent K(m) for correct dAMP incorporation. Thus, Arg(72) interactions with substrate are critical for the stability of the correct T.dAMP base pair when the 5'-CTGG sequence is present in the binding pocket for the nascent base pair. Collectively, the data show that a mutant polymerase may yield higher than normal average replication fidelity, yet paradoxically place specific sequences at very high risk of mutation.

Trypanosoma brucei is the causative agent for African sleeping sickness. We have made in vitro and in vivo studies on the allosteric regulation of the trypanosome ribonucleotide reductase, a key enzyme in the production of dNTPs needed for DNA synthesis. Results with the isolated recombinant trypanosome ribonucleotide reductase showed that dATP specifically directs pyrimidine ribonucleotide reduction instead of being a general negative effector as in other related ribonucleotide reductases, whereas dTTP and dGTP directed GDP and ADP reduction, respectively. Pool measurements of NDPs, NTPs, and dNTPs in the cultivated bloodstream form of trypanosomes exposed to deoxyribonucleosides or inhibited by hydroxyurea confirmed our in vitro allosteric regulation model of ribonucleotide reductase. Interestingly, the trypanosomes had extremely low CDP and CTP pools, whereas the dCTP pool was comparable with that of other dNTPs. The trypanosome ribonucleotide reductase seems adapted to this situation by having a high affinity for the CDP/UDP-specific effector dATP and a high catalytic efficiency, Kcat/Km, for CDP reduction. Thymidine and deoxyadenosine were readily taken up and phosphorylated to dTTP and dATP, respectively, the latter in a nonsaturating manner. This uncontrolled uptake of deoxyadenosine strongly inhibited trypanosome proliferation, a valuable observation in the search for new trypanocidal nucleoside analogues.

In order to understand the functional implication of residues constituting the dNTP-binding pocket of human immunodeficiency virus type 1 reverse transcriptase, we performed site-directed mutagenesis at positions 65, 72, 113, 115, 151, 183, 184, and 219, and the resulting mutant enzymes were examined for their biochemical properties and nucleotide selectivity on RNA and DNA templates. Mutations at positions 65, 115, 183, 184, and 219 had negligible to moderate influence on the polymerase activity, while Ala substitution at positions 72 and 151 as well as substitution with Ala or Glu at position 113 severely impaired the polymerase function of the enzyme. The K219A, Y115F, and Q151M mutants had no influence on the fidelity; Y183A, Y183F, K65A, and Q151N mutants exhibited higher fidelity on both RNA and DNA templates, while Y115A was less error-prone selectively on a DNA template. Analysis of the three-dimensional model of the enzyme-template primer-dNTP ternary complex suggests that residues Tyr-183, Lys-65, and Gln-151 may have impact on the flexibility of the dNTP-binding pocket by virtue of their multiple interactions with the dNTP, template, primer, and other neighboring residues constituting the pocket. Recruitment of the correct versus incorrect nucleotides may be a function of the flexibility of this pocket. A relatively rigid pocket would provide greater stringency, resulting in higher fidelity of DNA synthesis in contrast to a flexible pocket. Substitution of a residue having multiple interactions with a residue having reduced interaction capability will alter the internal geometry of the pocket, thus directly influencing the fidelity.

Hydroxyurea inhibits DNA synthesis by destroying the catalytically essential free radical of class I ribonucleoside diphosphate (rNDP) reductase, thereby blocking the de novo synthesis of deoxyribonucleotides. In mammalian cells, including those infected by vaccinia virus, hydroxyurea treatment causes a differential depletion of the four deoxyribonucleoside triphosphate pools, suggesting that the activities of rNDP reductase are differentially sensitive to hydroxyurea. In the presence of different substrates and allosteric modifiers, we measured rates of free radical destruction in the vaccinia virus-coded rNDP reductase, by following absorbance at 417 nm as a function of time after hydroxyurea addition. Also, we followed enzyme activity directly, by using a recently developed assay that allows simultaneous monitoring of the four activities, in the presence of substrates and effectors at concentrations that approximate the intracellular environment. We found the primary determinant of radical loss to be not the ensemble of allosteric ligands bound but the activity of the enzyme. Nucleoside triphosphate effectors accelerated radical decay, compared with rates seen with the free enzyme. Adding substrate to the holoenzyme, under conditions where the enzymatic reaction is proceeding, further accelerated radical decay. Alternative models are discussed, to account for selective depletion of purine nucleotide pools by hydroxyurea.

We have investigated the in vitro fidelity of Escherichia coli DNA polymerase III holoenzyme from a wild-type and a proofreading-impaired mutD5 strain. Exonuclease assays showed the mutD5 holoenzyme to have a 30-50-fold reduced 3'-->5'-exonuclease activity. Fidelity was assayed during gap-filling synthesis across the lacId forward mutational target. The error rate for both enzymes was lowest at low dNTP concentrations (10-50 microM) and highest at high dNTP concentration (1000 microM). The mutD5 proofreading defect increased the error rate by only 3-5-fold. Both enzymes produced a high level of (-1)-frameshift mutations in addition to base substitutions. The base substitutions were mainly C-->T, G-->T, and G-->C, but dNTP pool imbalances suggested that these may reflect misincorporations opposite damaged template bases and that, instead, T-->C, G-->A, and C-->T transitions represent the normal polymerase III-mediated base.base mispairs. The frequent (-1)-frameshift mutations do not result from direct slippage but may be generated via a mechanism involving "misincorporation plus slippage." Measurements of the fidelity of wild-type and mutD5 holoenzyme during M13 in vivo replication revealed significant differences between the in vivo and in vitro fidelity with regard to both the frequency of frameshift errors and the extent of proofreading.

The DNA cleavage-ligation reaction of DNA topoisomerase I was investigated employing synthetic DNA substrates containing 3'-deoxyadenosine or 3'-deoxythymidine at specific sites and acceptor oligonucleotides of different lengths. The modified nucleotides were substituted systematically within the putative enzyme-binding domain and also next to the high efficiency cleavage site to determine the effect of single base changes on enzyme function. Depending on the site of substitution, the facility of the cleavage and ligation reactions were altered. The bases at positions -1 and -2 on the noncleaved strand were found to be important for determining the site of cleavage. Inclusion of 3'-deoxythymidine in the scissile strand at position -1 permitted the demonstration that topoisomerase I can cleave and form a 2' --> 5'-phosphodiester linkage. Partial duplexes doubly modified at positions -4 or -6 in the noncleaved strand and at positions +1 or -1 within scissile strand were not good substrates for topoisomerase I, showing that cleavage can depend importantly on binding interactions based on structural alterations at spatially separated sites. Substitution of a 3'-deoxynucleotide on the scissile strand at position -6 enhanced formation of the ligation product resulting from cleavage at site 1 and suppressed cleavage at site 2.

The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addition according to a monoexponential time course. The pseudo-first order rate constant was determined for different concentrations of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast association of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate constants and dissociation equilibrium constants determined for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymatic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s-1 and a KS of 1-5 mM. The equilibrium dissociation constants for ADP, GDP, ddADP, and ddGDP were also determined by fluorescence titration of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3'-OH in ddNTP causes a 10-fold increase in KD. Contrary to "natural" NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in kcat values.

Propanodeoxyguanosine (PdG) is a model for several unstable exocyclic adducts formed by reaction of DNA with bifunctional carbonyl compounds generated by lipid peroxidation. The effect of PdG on DNA synthesis by human DNA polymerase beta was evaluated using template-primers containing PdG at defined sites. DNA synthesis was conducted in vitro and the products were analyzed by polyacrylamide gel electrophoresis and DNA sequencing. The extent of PdG bypass was low and the products comprised a mixture of base pair substitutions and deletions. Sequence analysis of all of the products indicated that the deoxynucleoside monophosphate incorporated "opposite" PdG was complementary to the base 5' to PdG in the template strand. These findings are very similar to recent results of Efrati et al. (Efrati, E., Tocco, G., Eritja, R., Wilson, S. H., and Goodman, M. F. (1997) J. Biol. Chem. 272, 2559-2569) obtained in DNA replication of template-primers containing abasic sites and suggest that PdG is a non-informational lesion when acted upon by polymerase (pol) beta. In addition to base pair substitutions and one- or two-base deletions, a four-base deletion was observed and the mechanism of its formation was probed by site-specific mutagenesis. The results indicated that this deletion occurred by one-base insertion followed by slippage to form a four-base loop followed by extension. All of the observations on pol beta replication of PdG-containing template-primers are consistent with a mechanism of lesion bypass that involves template slippage and dNTP stabilization followed by deoxynucleoside monophosphate incorporation and extension. This mechanism of PdG bypass is completely different than that previously determined for the Klenow fragment of DNA polymerase I and is consistent with recent structural models for DNA synthesis by pol beta.