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M J Modak

Publications and source records attributed to M J Modak.

At least 19 recordsLinked to original sources
PubMed1996

Biochemical analysis of catalytically crucial aspartate mutants of human immunodeficiency virus type 1 reverse transcriptase.

In order to clarify the role(s) of the individual member of the carboxylate triad in the catalytic mechanism of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, we carried out site-directed mutagenesis of D185, D186, and D110, followed by the extensive characterization of the properties of the individual mutant enzymes. We find that all three residues participate at or prior to the chemical step of bond formation. The incorporation pattern seen with phosphorothioate analogs of dNTP on both RNA-DNA and DNA-DNA template-primers indicated that D186 may be the residue that coordinates with the alpha-phosphate group of dNTP in the transition-state ternary complex. Further support for the role assigned to D186 was obtained by examination of the ability of the individual carboxylate mutants to catalyze the reverse of the polymerase reaction (pyrophosphorolysis). Mutants of D185 exhibited near-normal pyrophosphorolysis activity, while those of D186 were completely devoid of this activity. Thus, D185 appears to participate only in the forward reaction, probably required for the generation of nucleophile by interacting with the 3'-OH of the primer terminus, while D186 seems to be involved in both the forward and the reverse reactions, presumably by participating in the pentavalent intermediate transition state. Lack of any elemental effects during polymerization with mutant enzymes of residue D110, together with their inability to catalyze pyrophosphorolysis, suggest its probable participation in the metal-coordinated binding to the beta-gamma-phosphate of dNTP or PPi in the forward and reverse reactions, respectively. A molecular model of the ternary complex based on these results is also presented.

Aspartic Acid↗
PubMed1996

Significance of the O-helix residues of Escherichia coli DNA polymerase I in DNA synthesis: dynamics of the dNTP binding pocket.

In order to identify functionally important residues in the O and O1 helices of Escherichia coli DNA polymerase I, we mutated 9 residues of this region to alanine. The alanine substitutions result in moderate to severe effects on the polymerase activity of the individual mutant enzymes. Severe loss of activity is associated with R754A, K758A, F762A, and Y766A. However, the loss of polymerase activity with different template primers exhibited a rather unique pattern implying differential participation of the individual residue in the synthesis directed by poly(rA), poly(dA), and poly(dC) templates. The ability of all mutants to form E-DNA binary complex was found to be unaffected with the exception of Y766A and F771A, where significant reduction in the cross-linking of both the template and the primer strand was noted. Most interestingly, the catalytic activity of all inactive mutant enzymes, with the exception of K758A, could be restored by substituting Mn2+ in place of Mg2+ as a divalent cation. Based on these results and associated changes in the kinetic parameters and other properties of the individual mutant enzyme, we conclude the following: (a) Tyr 766 and Phe 771 are either involved in the binding of template-primer or are in the vicinity of the DNA binding track. (b) Residues Arg 754, Lys 758, Phe 762, and Tyr 766 appear to be required for the binding of Mg.dTTP, while only Arg 754 and Lys 758 are utilized in the polymerization of Mn.dTTP. (c) In the polymerization of dGTP, only Lys 758 appears essential regardless of the type of divalent cation. (d) Phe 762 participates only in the binding of Mg.dTTP. Finally, (e) based on the analysis of the time course of nucleotide incorporation, processivity, and pyrophosphorolysis reaction, we suggest that Lys 758 is probably involved in a conformational change of the ternary complexes preceding and following the chemical step. In summary, our results suggest that the formation of the dNTP binding pocket is a dynamic process which requires the participation of different residues depending on the type of dNTP and the divalent cation.

Base Sequence↗
PubMed1996

Role of methionine 184 of human immunodeficiency virus type-1 reverse transcriptase in the polymerase function and fidelity of DNA synthesis.

Methionine 184 of HIV-1 RT is a constituent of the catalytically crucial and highly conserved YXDD motif in the reverse transcriptase class of enzymes. We investigated the role of this residue by substituting it with Ala and Val by site-directed mutagenesis followed by extensive characterization of the two mutant enzymes. The kinetic parameters governing DNA synthesis directed by RNA and DNA templates indicated that both M184A and M184V mutants are catalytically as efficient as the wild type enzyme. Photoaffinity labeling of both the mutant and the wild type enzyme exhibited an identical affinity for RNA-DNA and DNA-DNA template primers. We further demonstrate that M-->V substitution at 184 position significantly increases the fidelity of DNA synthesis while M-->A substitution results in a highly error-prone enzyme without having compromised its efficiency of DNA synthesis. The M184V mutant exhibited a 25-45-fold increase in mismatch selectivity (ratio of k(cat)/K(m) of correct versus incorrect nucleotides) as compared to the WT enzyme. This pattern of error-prone synthesis is also confirmed by examining the abilities of the enzyme-(template-primer) covalent complexes to incorporate correct versus incorrect nucleotide onto the immobilized template-primer. The nature of error-prone synthesis by the M184A mutant shows an increase in both the mismatch synthesis and extension of the mismatched primer termini. Using a three-dimensional molecular model of the ternary complex of HIV-1 RT, template-primer, and dNTP, we observe that the strategic location of M184 may allow it to interact with the sugar moiety of either the primer nucleotide or the dNTP substrate.

Amino Acid Sequence↗
PubMed1995

Site-directed mutagenesis of arginine 72 of HIV-1 reverse transcriptase. Catalytic role and inhibitor sensitivity.

In order to determine the catalytic role of Arg72 of HIV-1 reverse transcriptase (RT), we carried out site-directed mutagenesis at codon 72. Two mutant proteins (R72A and R72K) were purified and characterized. With Arg to Ala substitution the kcat of the polymerase reaction was reduced by nearly 100-fold with poly(rA) template, but only about 5-15-fold with poly(rC) and poly(dC) templates. The Arg to Lys substitution exhibited a qualitatively similar pattern, although the overall reduction in kcat was less severe. Most interestingly, we noted a large difference in the rate constant of the first and second nucleotide incorporation by R72A, suggesting that Arg72 participates in the reaction after the formation of the first phosphodiester bond. We propose this step to be the pyrophosphate binding and removal step following the nucleotidyltransferase reaction. Support for this proposal is obtained from the observation that the R72A mutant (i) exhibited a pronounced translocation defect in the processivity analysis, (ii) lacked the ability to catalyze pyrophosphorolysis, and (iii) showed complete resistance to phosphonoformate, an analog of PPi.Arg72 is the first residue of HIV-1 RT proposed to be involved in the pyrophosphate binding/removal function of RT.

Arginine↗
PubMed1995

Glutamine 151 participates in the substrate dNTP binding function of HIV-1 reverse transcriptase.

In order to define the role of Gln151 in the polymerase function of HIV-1 RT, we carried out site-directed mutagenesis of this residue by substituting it with a conserved (Q151N) and a nonconserved residue (Q151A). Q151N exhibited properties analogous to those of the wild-type enzyme, while Q151A has severely impaired polymerase activity. The Q151A mutant exhibited a 15-100-fold reduction in kcat with RNA [poly(rC) and poly(rA)] templates, while only a 5-fold reduction could be seen with the DNA [poly(dC)] template. Most interestingly, the affinity of the Q151A mutant for dNTP substrate remained unchanged with RNA templates, but a significant increase in Km was noted with the DNA template. The binding affinity of Q151A for DNA remained unchanged, as judged by photoaffinity cross-linking. However, unlike the wild-type enzyme, the Q151A mutant failed to catalyze the nucleotidyl transferase reaction onto the primer terminus of the covalently immobilized template-primer. The enzyme showed profoundly altered divalent cation preference from Mg2+ to Mn2+. These results strongly implicate Q151 of HIV-1 RT in the substrate dNTP binding function and possibly in the following chemical (catalytic) step. The effects of the mutation seem to be through Q151 of the p66 catalytic subunit, as p66WTt/p51Q151A retains the wild-type kinetic constants and nucleotidyl transferase activity. In contrast, p66Q151A/p51WT is indistinguishable from Q151A (mutated in both subunits). A model of the ternary complex (enzyme-template-primer and dNTP) has been used to infer the possible mode by which Q151 may interact with the base moiety of the substrate as well as with Arg72, a residue present within the active site of HIV-1 RT.

Amino Acid Sequence↗
PubMed1994

Properties of tyrosine 766-->serine mutant of Escherichia coli DNA polymerase I: template-specific effects.

In order to determine the role of Tyr 766 of Escherichia coli DNA polymerase I in the catalysis of DNA synthesis, we investigated the properties of a Tyr 766-->Ser (Y766S) mutant of the Klenow fragment of E. coli DNA polymerase I. We found that the rates of incorporation of only dTTP but not the other dNTP substrates were affected in the reactions catalyzed by the mutant enzyme, when homopolymeric template-primers were used. The mutant enzyme exhibited a reduced rate of synthesis only with poly(rA)- or poly(dA)-directed reactions. Examination of the ability of the mutant and the wild-type enzymes to bind to dGTP and dTTP, as judged by UV-mediated cross-linking, indicated nearly identical binding efficiencies of both nucleotides. However, the ability of the mutant enzyme to bind to poly(rA).(dT)15 and poly(dA).(dT)15 was found to be significantly reduced as compared to the binding to heteropolymeric DNA. In order to further define the nature of template-mediated restriction on the catalytic activity of the mutant enzyme, its ability to copy DNA templates containing a stretch of AAAAA and ACACA sequences was compared. The results show that DNA synthesis catalyzed by the mutant enzyme is significantly retarded when it encounters the AAAAA region of the template but not the ACACA region. Product analysis of the reaction directed by the two template-primers showed that the mutant enzyme stalls/terminates synthesis upon encountering an AAAAA sequence in the template.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Photoaffinity labeling of DNA template-primer binding site in Escherichia coli DNA polymerase I. Identification of involved amino acids.

We have used two self-annealing template-primers (TPs) to covalently cross-link the Klenow fragment of Escherichia coli DNA polymerase I in its polymerase mode. The specificity of cross-linking is demonstrated by the observation that other template-primers, but not the template or primer alone, readily compete with self-annealing TPs. The enzyme-TP covalent complex is catalytically active and can incorporate one nucleotide on the primer terminus of the immobilized template-primer. Using a peptide mapping approach, we have identified a 17-amino acid tryptic peptide spanning residues 759-775 as a major constituent of the TP binding domain. Amino acid sequence analysis further revealed that Ile-765, Tyr-766 in the O-helix and Ser-769, Phe-771 in the O1-helix of the three-dimensional crystal structure of the Klenow fragment constitute the attachment site for TP.

Affinity Labels↗
PubMed1994

Purification and nucleic acid binding properties of a fragment of type C1/C2 heterogeneous nuclear ribonucleoprotein from thymic nuclear extracts.

A single-strand nucleic acid binding protein (C/F) that has an apparent molecular weight of 12,000 on SDS-polyacrylamide gel electrophoresis and that was originally thought to be the 12-kDa alpha-subunit of the AB form of terminal deoxynucleotidyl transferase (TdT) from calf thymus has been purified and identified as a fragment of the type C1/C2 hnRNP proteins. On the basis of NH2-terminal sequencing and mass spectrometric analysis, C/F contains approximately 94 residues and spans from residue 9 to approximately residue 102 in the type C1/C2 hnRNP proteins. C/F is presumably produced in vitro via limited proteolysis of the type C1/C2 hnRNP proteins following cell disruption. Since C/F corresponds almost exactly to the approximately 90-residue conserved ribonucleoprotein binding domain (RBD) that is shared by many eukaryotic RNA binding proteins, it provided an opportunity to better characterize the domain structure of the type C1/C2 hnRNP proteins and to compare the nucleic acid binding properties of the type C1/C2 and A1 [see Shamoo et al. (1994) Biochemistry, preceding paper in this issue] RNA binding domains. Like the type A1 RBD, the type C1/C2 RBD has an apparent occluded site size of 6-7 nucleotides. The type C1/C2 RBD binds non-cooperatively to homopolynucleotides and has preferential affinity for RNA and for single as opposed to double-stranded nucleic acids. The type C1/C2 RBD has about a 100-fold higher affinity than the type A1 RBD does for RNA and some of this increased affinity results from additional ionic interactions. The latter account for approximately 50% of the free energy of binding of the type C1/C2 RBD. While the type C1/C2 hnRNP proteins exist in vivo as a very tight tetramer with the structure (C1)3C2 [Barnett et al. (1989) Mol. Cell. Biol. 9, 492-498], the isolated type C1/C2 RBD is a monomer. Hence, the determinants for tetramerization appear to lie outside the type C1/C2 RBD. Phenylalanine 19 was identified as the only point of photochemical cross-linking of the type C1/C2 RBD to [d(T)]8. This residue corresponds to the major site of cross-linking of the A1 RBD to [d(T)]8 [Merrill, B. M., Stone, K. L., Cobianchi, F., Wilson, S. H., & Williams, K. R. (1988) J. Biol. Chem. 263, 3307-3313].(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Role of lysine 758 of Escherichia coli DNA polymerase I as assessed by site-directed mutagenesis.

Lys-758 of Escherichia coli DNA polymerase I has been implicated in the process of substrate dNTP binding (Basu, A., and Modak, M. J. (1987) Biochemistry 26, 1704-1709). To confirm and define the role of Lys-758 in the catalytic mechanism, we carried out site-directed mutagenesis of this residue. Catalytic activity of the purified mutant enzymes, K758A and K758R, showed severe reduction in the polymerase activity but little difference in the 3'-->5' exonuclease activity. Most interestingly, the catalytic ability of both mutant enzymes was maximally affected (300-1,000-fold decrease in kcat) with poly(dA).(dT)15 as template-primer (TP), whereas the ability to use poly(dC) templates decreased by only 20-fold in K758A and remained nearly unchanged with K758R. Kinetic characterization showed that Km(dNTP) increased moderately only with K758A, whereas Kd(TP) remained unchanged for both the mutants. However, binary complex formation between K758A and dNTP, but not between K758A and TP, was severely reduced. Analysis of the processive mode of DNA synthesis by K758A indicated that the mutant enzyme pauses at dA bases but does not dissociate from TP, suggesting a defect in its translocation ability. Thus, Lys-758 in polymerase I appears to participate in two distinct functions: (a) it facilitates the dNTP binding, and (b) it is required for the translocation along the template polynucleotide.

Base Sequence↗
PubMed1994

A computer-assisted analysis of conserved residues in the three-dimensional structures of the polymerase domains of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase.

Using a computer-assisted molecular modeling protocol, we have completed the three-dimensional structures of HIV-1 reverse transcriptase and the Klenow fragment of DNA polymerase I based on the C alpha crystal coordinates of the individual enzymes. The two model-built structures were then used to compare the electrostatic potential contours and analyze the spatial positions of residues conserved in the catalytic domains of the two enzymes. In spite of rather weak sequence similarity and different folding patterns between the DNA-dependent DNA polymerase (pol I) and the RNA-dependent DNA polymerases (RT), we have noted the occurrence of identical or similar residues at common spatial positions in pol I and RT in a three-dimensional context. The homologous residues present at equivalent spatial position in the Klenow fragment and the p66 subunit of HIV-1 RT may therefore imply their functional similarity. Furthermore, these conserved residues may represent a similar structure-function feature in all polymerases.

Amino Acid Sequence↗
PubMed1994

Identification and analysis of a template-primer (ds-DNA) binding cleft in E. coli DNA polymerase I: an electrostatic potential contour pattern of the modeled structure.

In the modeled structure of the Klenow fragment of E. coli DNA polymerase I, we have identified a distinct region that exhibits a strong electropositive potential contour. The examination of the distribution of the electropositive and negative potential across the two-dimensional slices of the modeled structure revealed that the positive potential was concentrated around the cleft. The approximate size and shape of the region appears well suited to accommodate eight base pairs of duplex DNA and is consistent with the position of the dsDNA binding cleft reported in the crystal structure [Beese et al., Science (1993) 260, 352-355].

Binding Sites↗
PubMed1993

Sulphydryl groups in the template-primer-binding domain of murine leukaemia virus reverse transcriptase. Identification and functional analysis of cysteine-90.

Treatment of murine leukaemia virus reverse transcriptase with benzophenone 4-maleimide inactivates DNA polymerase activity, but has no effect on the RNAase H function. Kinetic measurements indicated that benzophenone 4-maleimide is a competitive inhibitor with respect to template-primer binding, but is non-competitive with respect to dNTP binding. Enzyme modified with benzophenone 4-maleimide cannot bind template-primer or primer alone, as judged by u.v.-mediated cross-linking of radiolabelled substrates. Of the eight cysteine residues in murine leukaemia virus reverse transcriptase, only two were modified by benzophenone 4-maleimide, which were identified as Cys-90 and Cys-310 by comparative tryptic-peptide mapping and amino acid composition analysis. Inclusion of template-primer or primer alone in the modification mixture protected only Cys-90 from modification by benzophenone 4-maleimide. To investigate the role of Cys-90 in detail, we converted it to alanine by site-directed mutagenesis. The mutant enzyme, however, exhibited no loss either of DNA polymerase or of RNAase H activity. These results indicate that Cys-90 is located in a domain of murine leukaemia virus reverse transcriptase that binds template-primer, but may not have a direct role in the enzymic function of the enzyme. Ala-90 mutant murine leukaemia virus reverse transcriptase is at least 10-fold more susceptible to heat inactivation than is the wild-type enzyme, which suggests that Cys-90 in murine leukaemia virus reverse transcriptase may play a role in maintaining structural integrity.

Base Sequence↗
PubMed1993

Site directed mutagenesis of DNA polymerase I (Klenow) from Escherichia coli. The significance of Arg682 in catalysis.

We have reported that a domain containing Arg682 in the Klenow fragment of Escherichia coli DNA polymerase I (pol I) is important for the template-dependent dNTP-binding function [Pandey, V.N., Kaushik, N. A., Pradhan, D. S. & Modak, M. J. (1990) J. Biol. Chem. 265, 3679-3884]. In order to further define the role of Arg682 in the catalytic process, we have performed site-directed mutagenesis of this residue. For this purpose the Klenow-coding region of the DNA-pol-I gene was selectively amplified from the genomic DNA of E. coli and was cloned in an expression vector, pET-3a. This clone under appropriate conditions overproduces the Klenow fragment in E. coli. Using this clone (pET-3a-K) as the template, two mutant polymerase clones were constructed in which arginine has been replaced with either alanine, [R682A] pol I, or lysine [R682K] pol I. Both mutant enzymes showed significantly lower specific activity as compared to the wild-type enzyme. The kinetic analyses of the mutant enzymes indicated a 3-4-fold increase in the Km for the substrate dNTP, a 20-25-fold decrease in the Vmax and an overall decrease in the processive nature of DNA synthesis in both the mutant enzymes. The reverse mutation of Ala682 to the wild-type form Arg682 fully restored the processive nature and the polymerase activity of the enzyme. These observations suggest that the positively charged guanidino group in the side chain of Arg682 is catalytically important but not absolutely essential for synthesis of DNA. Furthermore it appears to maintain high processivity of the DNA synthesis catalyzed by the enzyme.

Arginine↗
PubMed1992

Structure-activity analyses of HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase is a dimeric enzyme which can exist in both homodimeric (p66/p66) and heterodimeric (p66/p51) forms. The monomeric subunits are catalytically inert. However, during DNA synthesis by the dimeric enzyme, only one subunit (p66) appears to carry out the catalysis, while the second subunit serves only a supportive role. In the case of the p66/p66 homodimers, we find that both the subunits are catalytically competent as judged by the observation that a) primer binding occurs to both subunits and b) catalytically inert dimers can be partially activated by replacement of one of the two inactive p66 subunits.

Acquired Immunodeficiency Syndrome↗
PubMed1992

A molecular model of the complete three-dimensional structure of the Klenow fragment of Escherichia coli DNA polymerase I: binding of the dNTP substrate and template-primer.

A complete three-dimensional structure of the Klenow fragment of Escherichia coli DNA polymerase I (pol I) has been proposed on the basis of molecular modeling and molecular mechanics studies using available C alpha coordinates. The structure seems quite reliable because the overall surface of electrostatic potentials calculated for the molecularly modeled enzyme closely resembles that reported for the X-ray structure. The modeled structure is then used in developing a ternary complex of dTTP and (dA)25-(dT)14 poised in its active site. The orientation of both substrates in the ternary complex was primarily guided by the amino acid residues which had been known to interact with dNTP and DNA substrates from earlier studies. The proposed model (a) explains the geometrical and physicochemical relationship of the two substrates with the various critical amino acid residues involved in the binding process and (b) suggests possible roles for additional residues in the binding and/or polymerization reaction. Furthermore, the ternary complex appears to satisfy many biochemical and genetic data concerning catalytic requirements known to exist for the polymerization reaction.

Amino Acid Sequence↗
PubMed1992

Identification of the primer binding domain in human immunodeficiency virus reverse transcriptase.

We have labeled the primer binding domain of HIV1-RT with 5'-32P-labeled (dT)15 primer using ultraviolet light energy. The specificity of the primer cross-linking to HIV1-RT was demonstrated by competition experiments. Both synthetic and natural primers, e.g., p(dA)15, p(dC)15, and tRNA(Lys), inhibit p(dT)15 binding and cross-linking to the enzyme. The observed binding and cross-linking of the primer to the enzyme were further shown to be functionally significant by the observation that tRNA(Lys) inhibits the polymerase activity on poly(rA).(dT)15 template-primer as well as the cross-linking of p(dT)15 to the enzyme to a similar extent. At an enzyme to p(dT)15 ratio of 1:3, about 15% of the enzyme can be cross-linked to the primer. To identify the domain cross-linked to (dT)15, tryptic peptides were generated and purified by a combination of HPLC on a C-18 reverse-phase column and DEAE-Sephadex chromatography. A single peptide cross-linked to p(dT)15 was identified. This peptide corresponded to amino acid residues 288-307 in the primary sequence of HIV1-RT as judged by amino acid composition and sequence analyses. Further, Leu(289)-Thr(290) and Leu(295)-Thr(296) of HIV1-RT appear to be the probable sites of cross-linking to the primer p(dT)15.

Amino Acid Sequence↗