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Deoxynucleotide triphosphate triphosphyohydrolyases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools by hydrolyzing dNTPs into deoxynucleosides and inorganic triphosphate (PPPi). While the vast majority of these enzymes display broad activity towards canonical dNTPs, exemplified by Sterile Alpha Motif (SAM) and Histidine-aspartate (HD) domain-containing protein 1 (SAMHD1), which blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels,Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric E. coli dGTPase. To obtain these structures, we applied UV-fluorescence microscopy, video analysis and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly-located on fixed target holders, resulting in the highest indexing-rates observed for a serial femtosecond crystallography (SFX) experiment. The structure features a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo form. Moreover, despite no sequence homology, dGTPase and SAMHD1 share similar active site and HD motif architectures; however, dGTPase residues at the end of the substrate-binding pocket mimic Watson Crick interactions providing Guanine base specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures sheds light into the mechanism by which long distance binding (25 Å) of single stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.Significance StatementdNTPases play a critical role in cellular survival through maintenance of cellular dNTP. While dNTPases display activity towards dNTPs, such as SAMHD1 –which blocks reverse transcription of HIV-1 in macrophages– Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. Here we use novel free electron laser data collection to shed light into the mechanisms of (Ec)-dGTPase selectivity. The structure features a dynamic active site where conformational changes are coupled to dGTP binding. Moreover, despite no sequence homology between (Ec)-dGTPase and SAMHD1, both enzymes share similar active site architectures; however, dGTPase residues at the end of the substrate-binding pocket provide dGTP specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP.

Human DNA polymerase η (Pol η) plays an essential protective role against skin cancer caused by cyclobutane thymine-thymine dimers (TTDs), a frequent form of DNA damage arising from exposure to the sun. This enzyme rescues stalled replication forks at the TTDs by inserting bases opposite these DNA defects. Herein we calculate binding free energies for a free deoxyribose nucleotide triphosphate, dATP or dGTP, to Pol η complexed with undamaged or damaged DNA. The calculations indicate that the binding of dATP to the enzyme-DNA complex is thermodynamically favored for TTD-containing DNA over undamaged DNA, most likely because of more extensive hydrogen-bonding interactions between the TTD and the enzyme that hold the TTD more rigidly in place. The calculations also illustrate that dATP binding is thermodynamically favored over dGTP binding at both thymine positions of the TTD, most likely due to more persistent and stable hydrogen-bonding interactions between the TTD and dATP than between the TTD and dGTP. This free energy difference is slightly greater for binding at the 5' thymine position than at the 3' thymine position, presumably because of stabilization arising from the A:T base pair formed at the 3' position of the TTD in the previous step of Pol η function. All of these trends in binding free energies are consistent with experimental measurements of binding strength, fidelity, processivity, and overall efficiency. The insights gained from this analysis have implications for drug design efforts aimed at modifying the binding properties of this enzyme for improving cancer chemotherapy treatments.

EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.

SAMHD1 is a dGTP-activated deoxynucleoside triphosphate triphosphohydrolase (dNTPase) whose dNTPase activity has been linked to HIV/SIV restriction. The mechanism of its dGTP-activated dNTPase function remains unclear. Recent data also indicate that SAMHD1 regulates retrotransposition of LINE-1 elements. Here we report the 1.8-A crystal structure of homotetrameric SAMHD1 in complex with the allosteric activator and substrate dGTP/dATP. The structure indicates the mechanism of dGTP-dependent tetramer formation, which requires the cooperation of three subunits and two dGTP/dATP molecules at each allosteric site. Allosteric dGTP binding induces conformational changes at the active site, allowing a more stable interaction with the substrate and explaining the dGTP-induced SAMHD1 dNTPase activity. Mutations of dGTP binding residues in the allosteric site affect tetramer formation, dNTPase activity and HIV-1 restriction. dGTP-triggered tetramer formation is also important for SAMHD1-mediated LINE-1 regulation. The structural and functional information provided here should facilitate future investigation of SAMHD1 function, including dNTPase activity, LINE-1 modulation and HIV-1 restriction.

Replication of a genome from any organism must occur with minimal mistakes if the progeny are to remain viable. Replicative DNA polymerases perform this task, making less than one mistake per 106 base-pairs synthesized.(1–4) Fidelity is increased by an additional 100-fold if the polymerase has exonuclease capability.(5–7) The number of errors made during replication depends on how well a DNA polymerase (pol) can recognize a dNTP as either correct or incorrect for pairing opposite the templating base. Sometimes a DNA pol can circumvent the geometric constraints imposed by residues in the Nascent Base-pair binding Pocket (NBP) so that incorrect dNTPs can be incorporated albeit with low efficiency. Based on crystal structures and studies using fluorescent probes, selection of the correct dNTP most likely occurs prior to phosphoryl transfer.(8–16) Subsequent to dNTP binding (Scheme 1), the polymerase Fingers domain closes, aligning the reactive centers of the substrates, leading to rapid nucleotidyl transfer.(13, 17–19) The incorrect dNTP either fails to induce Fingers closing, or if the Fingers do close the result is an unstable, but potentially productive, ternary complex.(13, 20–21) Scheme I Pathway of dNTP binding and incorporation. EDn is the open complex, and FDn is the closed complex. DNA pols from different families, and even within families, differ greatly in their ability to resist insertion of incorrect dNTPs. The B family DNA pol from bacteriophage RB69 (RB69pol) maintains high fidelity even when it encounters damaged templates or templating base analogues.(22–28) Interestingly, upon substitution of Y567 with Ala in the NBP, the mutant RB69pol inserts incorrect dNTPs with efficiencies 102 to 103 fold greater than wild type (wt) pol.(29) Crystal structures of ternary complexes of the RB69pol Y567A mutant suggest that the reduction in base selectivity exhibited by this mutant was likely due to an increase in NBP flexibility, leading to stable conformations of the templating base even when paired opposite incorrect dNTPs, a situation that would otherwise be blocked in the wt enzyme.(15, 29) However, a fuller understanding of how increased flexibility of residues in the NBP affects base selectivity requires analysis of kinetic steps that occur prior to nucleotidyl transfer. For this purpose we employed the highly fluorescent 1,3-diaza-2-oxophenoxazine (tC°) to probe the pre-insertion steps before chemistry. As shown in Fig 1, tC° is a tricyclic cytosine analogue. When presents as the templating base, it can be used to follow isomerization of DNA pols from an open to a closed conformation in response to binding of an incoming dNTP.(12, 14) Furthermore, unlike 2-aminopurine (2AP), a well-characterized fluorescent adenine analogue, where fluorescence quenching is totally dependent on base stacking, quenching of tC° fluorescence depends on the formation of hydrogen bonds (HB) with the templating base.(30–34) Sandin et al reported that tC° base pairs exclusively with guanine (G) and caused minimal perturbations in the structure of duplex DNA.(35–36) Kuchta's lab has recently discovered that tC° triphosphate (dtC°TP) is preferentially inserted opposite templating G and A residues by both KF and human DNA pol alpha.(37) They speculated that the incorporation of dAMP opposite tC° and vice-versa was due to the propensity of tC° to tautomerize into its imino form (Fig. 1).(37) Figure 1 Pairing of guanine (G) and adenine (A) opposite tC°. The analogue pairs with adenine upon tautomerization to the imino form. We found that tC° fluorescence was quenched only when paired opposite dGTP in a wt RB69pol ternary complex, reflecting formation of a stable nascent base-pair.(15) Surprisingly, we found that the net equilibrium dissociation constant (Kd,net) for dGTP binding opposite tC° with the Y567A mutant, in the absence of chemistry, was reduced by more than 200 fold relative to the corresponding wt ternary complex. In addition, dATP was able to form a stable nascent base-pair with tC° only with the Y567A mutant. To gain further insight into why this mutant has increased affinity for dGTP and was permissive for dATP binding, we determined the crystal structures of the dGTP/tC° and dATP/tC°-containing ternary complexes of the RB69pol Y567A mutant at 1.9 A resolution. We found that incoming dATP formed two HBs with an imino-tautomerized form of tC°. However, stabilization of the dATP/tC° base-pair required: (i) movement of the tC° backbone sugar into the DNA minor groove; (ii) lateral shifting of residues A567 and G568 toward residue Y416; (iii) tilting of the tricyclic ring of tC° base to avoid a steric clash with L561. Together, these data suggest that flexibility of the NBP provided by replacement of Y567 with Ala, led to a more favorable isomerization step (higher k2 value compared to the k2 exhibited by wt pol, as shown in Scheme 1) resulting in an increase in substrate binding affinity.

Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli. The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane. No effects were seen due to hyperconcentration of protein at the membrane surface. The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4 degrees C with dissociation constants ranging from 0.1 to 80 microM. The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25 degrees C.

A comparison of proteins encoded by normal human ras genes and by mutant ras H or ras K genes activated in human carcinomas revealed no changes in subcellular localization, posttranslational modification, or guanine nucleotide binding associated with activation. Subcellular fractionation indicated that both normal and activated ras proteins were associated exclusively with the membrane fraction. Furthermore, both normal and activated ras proteins exhibited similar degrees of posttranslational acylation. The K D for dGTP binding was 1.0–2.2 × 10 −8 M, with no consistent differences between normal and activated ras proteins. In addition, a survey of 13 possible competing nucleotides revealed no differences in the specificity of nucleotide binding associated with ras gene activation. These results indicate that structural mutations which activate ras gene transforming activity do not alter the protein's known biochemical parameters and in particular do not affect the protein's intrinsic ability to bind guanine nucleotides.

Captan (N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide) was shown to bind to DNA polymerase I from Escherichia coli. The ratio of [14C] captan bound to DNA pol I was 1:1 as measured by filter binding studies and sucrose gradient analysis. Preincubation of enzyme with polynucleotide prevented the binding of captan, but preincubation of enzyme with dGTP did not. Conversely, when the enzyme was preincubated with captan, neither polynucleotide nor dGTP binding was blocked. The modification of the enzyme by captan was described by an irreversible second-order rate process with a rate of 68 +/- 0.7 M-1 s-1. The interaction of captan with DNA pol I altered each of the three catalytic functions. The 3'----5' exonuclease and polymerase activities were inhibited, and the 5'----3' exonuclease activity was enhanced. In order to study the 5'----3' exonuclease activity more closely, [3H]hpBR322 (DNA-[3H]RNA hybrid) was prepared from pBR322 plasmid DNA and used as a specific substrate for 5'----3' exonuclease activity. When either DNA pol I or polynucleotide was preincubated with 100 microM captan, 5'----3' exonuclease activity exhibited a doubling of reaction rate as compared to the untreated sample. When 100 microM captan was added to the reaction in progress, 5'----3' exonuclease activity was enhanced to 150% of the control value. Collectively, these data support the hypothesis that captan acts on DNA pol I by irreversibly binding in the template-primer binding site associated with polymerase and 3'----5' exonuclease activities. It is also shown that the chemical reaction between DNA pol I and a single captan molecule proceeds through a Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)

The binding of modifiers and substrates to the ribonucleotide reductase from Lactobacillus leichmannii has been studied with the use of enzyme highly purified by a modified preparative procedure. This monomeric enzyme represents a simpler system than classical allosteric enzymes since the study of allosterism, regulation, and conformational responses in the latter is complicated by interaction between subunits. Analysis of earlier kinetic results indicates that ribonucleotide substrates bind weakly to the regulatory site (KD for GTP, 4 m m ) compared with binding to the catalytic site (KD for GTP 0.24 m m ), and in so doing produce substrate activation. The apparent rate constant for product formation when GTP is not bound to the regulatory site is 31% of that observed when GTP occupies the regulatory site. Part of this effect is due to tighter binding of coenzyme when substrate is bound to the regulatory site, a result confirmed by direct measurement of adenosylcobalamin binding. The effect of modifiers such as dGTP which accelerate the reduction of specific substrates is also in part due to increased coenzyme binding as measured both kinetically and by equilibrium dialysis and ultrafiltration methods. The modifiers also seem to have an obligatory role in the enzyme mechanism. Without their presence neither formation of the radical-pair intermediate from adenosylcobalamin nor associated reactions (coenzyme degradation, coenzyme H-exchange with water) occur under the experimental conditions observed. When modifier concentration dependence for these reactions is compared with estimates of equilibrium constants for dissociation of modifiers and substrates from complexes with the regulatory and catalytic sites it appears likely that binding at the regulatory site is responsible. Calculation of coenzyme saturation under the experimental conditions indicates, however, that insignificant amounts of coenzyme are bound to the enzyme except when the regulatory site is occupied, and this effect adequately explains the necessity for modifiers. Binding of dGTP does not alter the circular dichroism of the enzyme in the far-ultraviolet region (200 to 250 nm) but causes significant changes in the aromatic region, and therefore appears to perturb tryptophyl and tyrosyl residues though not necessarily directly. Measurements have been made of the fractional change in sedimentation coefficient due to dGTP binding to the enzymes by the use of an adjustable Raleigh mask and double sector cell in the Beckman model E ultracentrifuge. Values for Δs/ s of −1.14% at 22° and −1.16% at 37° were obtained. The former, after correction for the fractional increase due to the calculated effect of dGTP on the buoyant weight of the protein, gives a value of (Δs/ s corr = −2.84% . If this is due to increase in hydrodynamic volume of the protein alone, it corresponds to an 8.8% increase. If it were entirely due to increased departure from spherical shape it would correspond to a change in axial ratio from 2.9 to 3.2. In the presence of saturating concentrations of ATP and dihydrolipoate and the maximum permissible concentration of a coenzyme analogue Δs/ s was −0.33%, the corrected value being about −1.99% at 37°. These conformational responses are as great as determined for multimeric enzymes such as in the effect of CTP on aspartate transcarbamylase, and are consistent with relay of the regulatory signal over a considerable intersite distance.