Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex.

Lesser, David; Grajkowski, Andrzej; Kurpiewski, Michael R.; Koziołkiewicz, Maria; Stec, Wojciech J.; Jen‐Jacobson, Linda

doi:10.1016/s0021-9258(18)35836-8

Cited by 60 publications

(44 citation statements)

References 30 publications

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“…The substitution by sulfur of a nonbridging diastereotopic oxygen at the α-phosphate of a dNTP introduces a chiral center at P α , with the replacement of the pro-R and pro-S oxygen atoms resulting in the formation of the R p -dNTPαS and S p -dNTPαS diastereomers containing S 1A –O 2A and O 1A –S 2A atoms respectively (Figure ). Given that the incorporation of R p and S p diastereomers into nucleotides and nucleic acids often results in differential properties with respect to the action of stereoselective enzymes and receptors, − we sought to test the ability of R p -dNTPαS and S p -dNTPαS analogues to support SAMHD1 tetramerization through binding at AL1 and AL2 and assess their properties as substrates or inhibitors at the SAMHD1 active site.…”

Section: Resultsmentioning

confidence: 99%

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of R_p- and S_p-dNTPαS Diastereomers

et al. 2021

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SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2′-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4+ myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the Rp and Sp diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in Sp-dNTPαS but not Rp-dNTPαS diastereomers prevents Mg2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that Sp-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while Rp-dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, Rp-dGTPαS, in which an Fe–Mg-bridging water species is poised for nucleophilic attack on the Pα. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the Sp-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of Rp-dNTPαS nucleotides and for a mode of competitive inhibition by Sp-dNTPαS nucleotides that competes with formation of the enzyme–substrate complex.

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Section: Resultsmentioning

confidence: 99%

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of R_p- and S_p-dNTPαS Diastereomers

et al. 2021

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“…Selected H-phosphonate derivatives have been used in one study (Jeltsch et al, 1993), but they are generally not stable to the conditions of DNA synthesis and deprotection (Zon & Stec, 1991). An alternative linkage that could alter the nature of the interactions to the phosphodiester linkage would be the chiral phosphorothioate (Figure 1c), and such chiral derivatives have been used in recent studies (Koziolkiewicz & Stec, 1992;Lesser et al, 1992;Kurpiewski et al, 1996;Thorogood et al, 1996). However, much of the charge associated with a phosphorothioate diester appears to be localized on the sulfur atom, and this functional group can in principle still participate in hydrogen bonding or ionic interactions.…”

Section: Resultsmentioning

confidence: 99%

“…A recent study with T4 endonuclease V (Iwai et al, 1994), and a second with the HIV Tat protein (Pritchard et al, 1994), both involved the preparation of DNA or RNA sequences, respectively, containing racemic methylphosphonates, followed by their resolution into stereochemically pure isomers for analysis of protein binding. Studies with the EcoRI restriction endonuclease have described the use of chiral phosphorothioates to probe essential interactions to the phosphates in the complex involving the GAATTC recognition site (Koziolkiewicz & Stec, 1992;Lesser et al, 1992;Kurpiewski et al, 1996). An earlier study with the lac repressor employed chiral methylphosphonate analogues (Noble et al, 1984).…”

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confidence: 99%

Probing Contacts to the DNA Backbone in the trp Repressor−Operator Sequence-Specific Protein−Nucleic Acid Complex Using Diastereomeric Methylphosphonate Analogues

Smith

McLaughlin

1997

Biochemistry

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Fourteen analogue DNA sequences containing the trp operator sequence and a single diastereomeric methylphosphonate linkage are each prepared from the stereochemically pure nucleoside methylphosphonate dimer building block, prepared as a phosphoramidite. The analogue sequences are shown to be single diastereomers on the basis of HPLC analysis of the digestion mixture; in each case, only a single diastereomeric dimer is present. These analogue sequences can be used effectively to probe for interactions to either of the prochiral phosphate oxygens as illustrated by their use to identify critical interactions in the trp repressor-operator complex. In a number of cases, the pairs of diastereomeric analogue sequences exhibit variable binding affinities that can be used to identify one of the prochiral phosphate oxygens as a critical site for complex-stabilizing interactions. Upon the basis of dissociation constants, apparent incremental binding energies are assigned to specific interactions. In all but one example, these identified sites for interactions to the phosphate backbone can be correlated with contacts implicated by the crystal structure analysis of the trp repressor-operator complex.

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“…Pure stereoisomers of the modified DNA strand were obtained in two steps. First, synthesis and separation of pure diastereoisomers of d [5'-DMT-ABzp(s)OEtT] were carried out as described earlier (Lesser et al, 1992). 31P NMR analysis had shown that diastereomeric purity of the Rp isomer was >96%, while the purity of the Sp isomer was 1 Abbreviations: ID NMR, one-dimensional NMR; 2D NMR, twodimensional NMR; 2D NOE, two-dimensional nuclear Overhauser effect; ATP, adenosine triphosphate; COSY, correlation spectroscopy; DQF-COSY, double-quantum filtered COSY; EDTA, ethylenediaminetetraacetate; FID, free induction decay; RT, retention time; -duplex, hybrid duplex d(GCTATAApsTGG)-r(CCAUUAUAGC) where the modified phosphate has an -type chirality; 5-duplex, hybrid duplex where the modified phosphate has an S-type chirality; TBAF, tetrabutylammonium fluoride; TEAA, triethylammonium; THF, tetrahydrofuran.…”

Section: Methodsmentioning

confidence: 99%

Structural Study of a DNA.cntdot.RNA Hybrid Duplex with a Chiral Phosphorothioate Moiety by NMR: Extraction of Distance and Torsion Angle Constraints and Imino Proton Exchange Rates

González

Stec²,

Kobylańska³

et al. 1994

Biochemistry

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The solution structure of the thiophosphate-modified DNA.RNA hybrid duplex d(GCTATAApsTGG).r(CCAUUAUAGC) has been studied by NMR. Two samples with pure stereochemistry in the modified phosphate have been investigated. Two-dimensional NMR (2D NMR) methods have been applied to assign nearly all the resonances in both duplexes. Scalar coupling constants have been determined by comparing quantitative simulations with experimental double-quantum filtered COSY (DQF-COSY) cross-peaks. More than 300 distance constraints have been obtained from two-dimensional nuclear Overhauser spectroscopy (2D NOE) spectra recorded in D2O and H2O by using a complete relaxation matrix analysis as implemented in the program MARDIGRAS. This hybrid duplex presents a heteronomous structure. Riboses in the RNA strand are found in a N-type conformation typical of the A-form family as shown by the lack of H1'-H2' cross-peaks in DQF-COSY spectra and confirmed by the measured interproton distances. In contrast, the DNA strand adopts a different conformation with sugar puckers partially in the S-type domain, which is not in agreement with the A-family of structures. Coupling constants in deoxyriboses are not consistent with any single sugar conformation. Therefore, sugar pucker pseudorotation parameters are calculated according to a two-state dynamic equilibrium between N- and S-type conformers. In general, the population of major S conformer is lower than in double-stranded DNA duplexes, indicating that hybrid duplexes may be more flexible than pure DNA or RNA. The only differences observed in the spectra between the two stereoisomers studied originate from resonances of protons located near the modified phosphate. No significant differences in interproton distance have been detected, and only a slight difference of sugar pucker in the 5' neighbor has been found. The sulfur atom appears to be well-accommodated without further changes in the structure of the hybrid.

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Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex.

Cited by 60 publications

References 30 publications

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of R_p- and S_p-dNTPαS Diastereomers

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of R_p- and S_p-dNTPαS Diastereomers

Probing Contacts to the DNA Backbone in the trp Repressor−Operator Sequence-Specific Protein−Nucleic Acid Complex Using Diastereomeric Methylphosphonate Analogues

Structural Study of a DNA.cntdot.RNA Hybrid Duplex with a Chiral Phosphorothioate Moiety by NMR: Extraction of Distance and Torsion Angle Constraints and Imino Proton Exchange Rates

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Stereoselective interaction with chiral phosphorothioates at the central DNA kink of the EcoRI endonuclease-GAATTC complex.

Cited by 60 publications

References 30 publications

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of Rp- and Sp-dNTPαS Diastereomers

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of Rp- and Sp-dNTPαS Diastereomers

Probing Contacts to the DNA Backbone in the trp Repressor−Operator Sequence-Specific Protein−Nucleic Acid Complex Using Diastereomeric Methylphosphonate Analogues

Structural Study of a DNA.cntdot.RNA Hybrid Duplex with a Chiral Phosphorothioate Moiety by NMR: Extraction of Distance and Torsion Angle Constraints and Imino Proton Exchange Rates

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Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of R_p- and S_p-dNTPαS Diastereomers

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of R_p- and S_p-dNTPαS Diastereomers