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PubMed2002

Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling.

Extracellular signals transduced via receptor tyrosine kinases, G-protein-coupled receptors or integrins activate Ras, a key switch in cellular signalling. Although Ras can activate multiple downstream effectors (PI3K, Ral em leader ) one of the major activated pathway is a conserved sequential protein kinase cascade referred to as the mitogen activated protein (MAP) kinase module: Raf>MEK>ERK. The fidelity of signalling among protein kinases and the spatio-temporal activation are certainly key determinants for generating precise biological responses. The fidelity is ensured by scaffold proteins, a sort of protein kinase "insulators" and/or specific docking sites among the members of the signalling cascade. These docking sites are found in upstream and downstream regulators and MAPK substrates [Nat Cell Biol 2 2000 110]. The duration and the intensity of the response are in part controlled by the compartmentalisation of the signalling molecules. Growth factors promote nuclear accumulation and persistent activation of ERK (p42/p44 MAP kinases) during the entire G1 period with an extinction during S-phase. These features are exquisitely well controlled by (i) the temporal induction of the MAP kinase phosphatases, MKP1-3, and (ii) the compartmentalisation of the signalling molecules. We have shown that MKP1-2 induction is strictly controlled by the activation of the MAP kinase module providing evidence for an autoregulatory mechanism. This negative regulatory loop was further enhanced by the capacity of ERK to phosphorylate MKP1 and 2. This action reduced the degradation rate of these MKPs through the ubiquitin-proteasomal system [Science 286 1999 2514]. Whereas the two upstream kinases of the module, Raf and MEK remained cytoplasmic, ERK anchored to MEK in the cytoplasm of resting cells, rapidly translocated to the nucleus upon mitogenic stimulation. This process was rapid, reversible, and controlled by the strict activation of the MAPK cascade. Prevention of this nuclear translocation, by overexpression of a cytoplasmic ERK-docking molecule (inactive MKP3) prevented growth factor-stimulated DNA replication [EMBO J 18 1999 664]. Following long term stimulation, ERK progressively accumulated in the nucleus in an inactive form. This nuclear retention relied on the neosynthesis of short-lived nuclear anchoring proteins. Nuclear inactivation and sequestration was likely to be controlled by MAP kinase phosphatases 1 and 2. Therefore we propose that the nucleus represents a site for ERK action, sequestration and signal termination [J Cell Sci 114 2001 3433]. In addition, with the generation of mice invalidated for each of the ERK isoforms, we will illustrate that besides controlling cell proliferation the ERK cascade also controls cell differentiation and cell behaviour [Science 286 1999 1374].

Animals↗
PubMed2002

poliota-dependent lesion bypass in vitro.

Based upon phylogenetic relationships, the broad Y-family of DNA polymerases can be divided into various subfamilies consisting of UmuC (polV)-like; DinB (polIV/polkappa)-like; Rev1-like, Rad30A (poleta)-like and Rad30B (poliota)-like polymerases. The polIV/polkappa-like polymerases are most ubiquitous, having been identified in bacteria, archaea and eukaryotes. In contrast, the polV-like polymerases appear restricted to bacteria (both Gram positive and Gram negative). Rev1 and poleta-like polymerases are found exclusively in eukaryotes, and to date, poliota-like polymerases have only been identified in higher eukaryotes. In general, the in vitro properties of polymerases characterized within each sub-family are quite similar. An exception to this rule occurs with the poliota-like polymerases, where the enzymatic properties of Drosophila melanogaster poliota are more similar to that of Saccharomyces cerevisiae and human poleta than to the related human poliota. For example, like poleta, Drosophila poliota can bypass a cis-syn thymine-thymine dimer both accurately and efficiently, while human poliota bypasses the same lesion inefficiently and with low-fidelity. Even in cases where human poliota can efficiently insert a base opposite a lesion (such as a synthetic abasic site, the 3'T of a 6-4-thymine-thymine pyrimidine-pyrimidone photoproduct or opposite benzo[a]pyrene diol epoxide deoxyadenosine adducts), further extension is often limited. Thus, although poliota most likely arose from a genetic duplication of poleta millions of years ago as eukaryotes evolved, it would appear that poliota from humans (and possibly all mammals) has been further subjected to evolutionary pressures that have "tailored" its enzymatic properties away from lesion bypass and towards other function(s) specific for higher eukaryotes. The identification of such functions and the role that mammalian poliota plays in lesion bypass in vivo, should hopefully be forthcoming with the construction of human cell lines deleted for poliota and the identification of mice deficient in poliota.

Animals↗
PubMed1995

Vaccinia virus DNA ligase: specificity, fidelity, and inhibition.

Vaccinia DNA ligase and mammalian DNA ligases II and III comprise a distinct subgroup of structurally homologous enzymes within the eukaryotic DNA ligase family. The specificity and fidelity of the viral enzyme were investigated using purified recombinant ligase and synthetic duplex DNA substrates containing a single strand discontinuity. Vaccinia ligase catalyzed efficient strand joining on nicked DNAs in the presence of magnesium and ATP (Km = 95 microM). dATP, ITP, AMPPCP, 3'dATP, and ATP alpha S could not substitute for ATP; of these, 3'dATP and ATP alpha S were inhibitors of ligation. The vaccinia enzyme was unable to seal strands across a 1 nt (nucleotide) or 2 nt gap. Ligase action at a 1 nt gap resulted in accumulation of high levels of the normally undetectable DNA-adenylate reaction intermediate. In contrast, no DNA-adenylate was formed at a 2 nt gap. A native gel mobility shift assay showed that vaccinia DNA ligase was capable of discriminating between nicked and gapped DNAs at the substrate binding step. The ligase was fairly tolerant of mismatches at a nick involving the 5' phosphate donor terminus but was inhibited strongly by mismatches at the 3' OH acceptor terminus, especially by purine.-purine mispairs. These findings underscore the importance of a proper 3' OH terminus in substrate recognition and reaction chemistry but also raise the possibility that ligase may generate mutations during DNA repair by sealing DNA molecules with mispaired ends. Ligase was inhibited by several DNA binding drugs, including, in order of decreasing potency, distamycin, ethidium bromide, and actinomycin. Strand joining by purified ligase was not affected by etoposide, a drug that inhibits vaccinia virus replication in vivo and which depends on the presence of vaccinia ligase for its antiviral action.

Adenosine Triphosphate↗
PubMed1996

Misincorporation by HIV-1 reverse transcriptase promotes recombination via strand transfer synthesis.

Genome heterogeneity in retroviruses derives from poor fidelity of the reverse transcriptase (RT) and recombination via RT-catalyzed strand transfer synthesis. RTs lack proofreading ability, and they proficiently extend primers with mismatched termini. Recombination reactions carried out in vitro are accompanied by a high frequency of base substitution errors, suggesting a relationship. Here we provide evidence that misincorporation during RNA-directed DNA synthesis promotes strand transfer recombination. Experiments involved measurement of DNA synthesis, RNase H-directed cleavage, and strand transfer synthesis from preformed mismatched primers on RNA templates by human immunodeficiency virus (HIV) RT in vitro. A significant pause in synthesis occurred from a G(primer). rA(template) mismatch compared to the synthesis from a correctly paired (T.A) primer. The misincorporation-induced pause allowed an unusually large area of RT-RNase H-directed cleavage of the template RNA beneath the primer. Strand transfer to an acceptor molecule with sequence identical to the template RNA was about 50% more efficient than if the primer had had a correctly paired terminus. Overall transfer was measured over a large region of homology. Assuming that enhanced transfer occurs primarily at the site of the mismatch, the actual increase in transfer at that site must have been 1-2 orders of magnitude. Inclusion of a different acceptor molecule with complete complementarity to the originally mismatched 3' primer terminus resulted in an additional 2-fold increase in strand transfer efficiency. Overall, these results suggest the mechanism by which misincorporation during minus strand DNA synthesis in retroviral replication would promote high frequency recombination.

DNA Primers↗
PubMed1985

2'-Fluoro-2'-deoxypolynucleotides as templates and inhibitors for RNA- and DNA-dependent DNA polymerases.

Poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl)) and poly(2'-deoxycytidylic acid) (poly(dCfl)) were tested as templates in DNA synthesis reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha, mouse cell DNA polymerase gamma and avian myeloblastis virus (AMV) reverse transcriptase. Poly(dAfl).(dT)12 can fully substitute for poly(rA).(dT)12 as template with DNA polymerase gamma, to 50% with reverse transcriptase, but was poorly recognized by DNA polymerase alpha. DNA synthesis by reverse transcriptase with poly(dCfl).(dG)12 as template was 50% of that with poly(rC).(dG).(dG)12. The use of 2'-fluoropolymers as templates was more efficient at 37 degrees C than at 25 degrees C. No appreciable differences on the fidelity of DNA synthesis by reverse transcriptase were observed when dCMP misincorporation was measured with poly(dAfl).(dT)12 or poly(rA).(dT)12 as template primers. Poly(C) and poly-2'-O-methylcytidylic acid had no significant effect on the reaction catalyzed by DNA polymerase gamma and reverse transcriptase, independent of the synthetic polynucleotide complex utilized as template. On the other hand, poly(dCfl) was an inhibitor when poly(rA).(dT)12 or poly(dA).(dT)12 were used as templates, but not when poly(dAfl).(dT)12 was employed. Analogous results have been obtained with activated DNA and AMV 70 S RNA as templates in the reverse transcriptase reaction. The inhibition by poly(dCfl) was noncompetitive with regard to TTP, poly(dA) and poly(rA). Xenopus laevis oocytes DNA polymerase alpha was not inhibited by poly(dCfl).

Animals↗
PubMed2006

Measuring the fidelity of translesion DNA synthesis.

A method is described to measure the fidelity of copying past a DNA lesion in a defined sequence on a synthetic oligonucleotide primer-template. The DNA product is the result of a complete lesion bypass reaction, i.e., containing all four deoxynucleotide triphosphates and requiring both insertion opposite the lesion and multiple extensions from the resulting primer termini containing the lesion. The nascent strand is recovered and hybridized to a gapped region of the lacZalpha complementation gene of the M13mp2 genome. When this DNA is introduced into Escherichia coli, errors made during translesion DNA synthesis are detected by M13 plaque colors. Sequencing of DNA from mutant plaques defines the types of errors and permits calculation of error rates for base substitutions, insertions, and deletions. The method is illustrated here for bypass of a cis-syn thymine-thymine dimer by human DNA polymerase eta. The assay can be used with other lesions in various sequence contexts and with other polymerases with or without accessory proteins.

Animals↗
PubMed1986

On the fidelity of DNA synthesis. Pyrophosphate-induced misincorporation allows detection of two proofreading mechanisms.

The effect of pyrophosphate on the fidelity of in vitro DNA synthesis has been examined. Pyrophosphate enhances misincorporation by Escherichia coli DNA polymerase I in copying phi X174 DNA. The increased misincorporation is directly proportional to the extent of inhibition of the rate of polymerization. In contrast, pyrophosphate is not detectably mutagenic with avian myeloblastosis virus DNA polymerase or DNA polymerases alpha and beta from animal cells, which lack associated proofreading activities. This suggests that increased misincorporation by pyrophosphate is not due to an increase in misinsertions by DNA polymerase, but rather due to inhibition of proofreading by pyrophosphate. However, the pyrophosphate-induced infidelity has a different specificity from, and is not competitive with, two experimental markers of 3'----5' exonuclease proofreading; i.e. the effects of the next nucleotide or the addition of deoxynucleoside monophosphates. These distinctive features suggest a second mode of proofreading susceptible to inhibition by pyrophosphate. This concept is discussed in relation to models for proofreading described in the literature.

Bacteriophage phi X 174↗
PubMed1989

Exonucleolytic proofreading by a mammalian DNA polymerase.

Porcine liver DNA polymerase gamma contains exonuclease activity capable of digesting DNA in the 3'----5' direction, releasing deoxyribonucleoside 5'-monophosphates. The exonuclease activity excises 3'-terminal bases from both matched and mismatched primer termini, with a preference for mismatched bases. Under polymerization conditions, mismatch excision by the exonuclease occurs prior to polymerization by polymerase gamma, and this excision can be inhibited by adding to the reaction a high concentration of dNTP substrates and/or nucleoside 5'-monophosphates. In an M13mp2-based reversion assay for detecting single-base substitution errors, porcine liver polymerase gamma is highly accurate; the estimated base substitution error rate is less than one error for each 500,000 bases polymerized. Lower fidelity is observed using reaction conditions that inhibit the exonuclease activity, strongly suggesting that the exonuclease proofreads errors during polymerization. However, in a forward mutation assay capable of detecting all 12 mispairs at a variety of template positions, certain base substitution errors are readily detected even using unperturbed polymerization conditions. Thus, for some errors, polymerase gamma is not highly accurate, suggesting that proofreading is not equally active against all mispairs. To examine if the polymerase and exonuclease activities are physically as well as functionally associated, both activities were monitored during purification by four procedures, each based on a different separation principle. The two activities copurify during chromatography using phosphocellulose, heparin-agarose, or double-strand DNA-cellulose, and during velocity sedimentation in a glycerol gradient containing 0.5 M KCl. These results suggest that the polymerase and exonuclease activities are physically associated. It remains to be determined if they reside in the same subunit.

Animals↗
PubMed1999

Base substitution specificity of DNA polymerase beta depends on interactions in the DNA minor groove.

To examine the hypothesis that interactions between a DNA polymerase and the DNA minor groove are critical for accurate DNA synthesis, we studied the fidelity of DNA polymerase beta mutants at residue Arg(283), where arginine, which interacts with the minor groove at the active site, is replaced by alanine or lysine. Alanine substitution, removing minor groove interactions, strongly reduces polymerase selectivity for all single-base mispairs examined. In contrast, the lysine substitution, which retains significant interactions with the minor groove, has wild-type-like selectivity for T.dGMP and A.dGMP mispairs but reduced selectivity for T.dCMP and A.dCMP mispairs. Examination of DNA crystal structures of these four mispairs indicates that the two mispairs excluded by the lysine mutant have an atom (N2) in an unfavorable position in the minor groove, while the two mispairs permitted by the lysine mutant do not. These results suggest that unfavorable interactions between an active site amino acid side chain and mispair-specific atoms in the minor groove contribute to DNA polymerase specificity.

Alanine↗
PubMed2002

Threonine 79 is a hinge residue that governs the fidelity of DNA polymerase beta by helping to position the DNA within the active site.

DNA polymerase beta (pol beta) is an ideal system for studying the role of its different amino acid residues in the fidelity of DNA synthesis. In this study, the T79S variant of pol beta was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol beta and has no contact with either the DNA template or the incoming dNTP substrate. The T79S protein produced 8-fold more multiple mutations in the herpes simplex virus type 1-thymidine kinase assay than wild-type pol beta. Surprisingly, T79S is a misincorporation mutator only when using a 3'-recessed primer-template. In the presence of a single nucleotide-gapped DNA substrate, T79S displays an antimutator phenotype when catalyzing DNA synthesis opposite template C and has similar fidelity as wild type opposite templates A, G, or T. Threonine 79 is located directly between two helix-hairpin-helix motifs located within the 8-kDa and thumb domains of pol beta. As the pol beta enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90 degrees bend of the DNA. The function of the bent DNA is to present the templating base to the incoming nucleotide substrate. We propose that Thr-79 is part of a hydrogen bonding network within the helix-hairpin-helix motifs that is important for positioning the DNA within the active site. We suggest that alteration of Thr-79 to Ser disrupts this hydrogen bonding network and results in an enzyme that is unable to bend the DNA into the proper geometry for accurate DNA synthesis.

Base Sequence↗
PubMed2007

The origin recognition complex functions in sister-chromatid cohesion in Saccharomyces cerevisiae.

High-fidelity chromosomal segregation requires the properly timed establishment of sister-chromatid cohesion mediated by the Cohesin complex, and its resolution at the metaphase-to-anaphase transition. We have examined cell-cycle progression in a yeast strain from which the origin recognition complex protein Orc2 was depleted after the assembly of prereplication complexes. We find that Orc2 depletion causes a delay in progression through mitosis, reflecting activation of both the DNA-damage and Mad2-spindle checkpoints. Surprisingly, sister-chromatid cohesion is impaired in Orc2-depleted cells, although Cohesin subunits are properly associated with chromatin. Reexpression of Orc2 in late G2/M phase restores chromatid cohesion. Finally, the targeting of Orc2 to a specific chromosomal locus suppresses premature sister-chromatid separation locally in a temperature-sensitive cohesin mutant. We conclude that ORC mediates sister-chromatid interaction on a pathway that is additive with Cohesin-mediated pairing.

Acetyltransferases↗
PubMed1990

Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage.

In eucaryotic cells, incompletely replicated or damaged chromosomes induce cell cycle arrest in G2 before mitosis, and in the yeast Saccharomyces cerevisiae the RAD9 gene is essential for the cell cycle arrest (T.A. Weinert and L. H. Hartwell, Science 241:317-322, 1988). In this report, we extend the analysis of RAD9-dependent cell cycle control. We found that both induction of RAD9-dependent arrest in G2 and recovery from arrest could occur in the presence of the protein synthesis inhibitor cycloheximide, showing that the mechanism of RAD9-dependent control involves a posttranslational mechanism(s). We have isolated and determined the DNA sequence of the RAD9 gene, confirming the DNA sequence reported previously (R. H. Schiestl, P. Reynolds, S. Prakash, and L. Prakash, Mol. Cell. Biol. 9:1882-1886, 1989). The predicted protein sequence for the Rad9 protein bears no similarity to sequences of known proteins. We also found that synthesis of the RAD9 transcript in the cell cycle was constitutive and not induced by X-irradiation. We constructed yeast cells containing a complete deletion of the RAD9 gene; the rad9 null mutants were viable, sensitive to X- and UV irradiation, and defective for cell cycle arrest after DNA damage. Although Rad+ and rad9 delta cells had similar growth rates and cell cycle kinetics in unirradiated cells, the spontaneous rate of chromosome loss (in unirradiated cells) was elevated 7- to 21-fold in rad9 delta cells. These studies show that in the presence of induced or endogenous DNA damage, RAD9 is a negative regulator that inhibits progression from G2 in order to preserve cell viability and to maintain the fidelity of chromosome transmission.

Cell Cycle↗
PubMed1990

DNA polymerase alpha-primase complexes from carcinogen-treated Chinese hamster ovary cells.

In order to investigate whether carcinogens induce alterations of the DNA polymerase alpha-primase complex we compared the physiochemical and catalytic properties and the fidelity of DNA synthesis of DNA polymerase alpha-primase complexes from carcinogen-treated and untreated Chinese hamster ovary cells. Complexes were purified by ion exchange or by immunoaffinity chromatography and both DNA-polymerizing activities and those of ancillary enzymes, such as RNA primase and exonuclease, were examined. Further characterization of the complexes included determination of the relative molecular masses, sedimentation coefficients, and diffusion coefficients, and measurements of the KmS for deoxynucleotide triphosphates and DNA templates, which were identical for the preparations from both carcinogen-treated and untreated cells. The fidelity of DNA polymerase alpha-primase complexes measured by the phi X174am3 reversion assay was also similar in carcinogen-treated and untreated cells. Thus, a carcinogen-mediated induction of a DNA polymerase alpha-primase complex with low fidelity was not observed within the detection limits of the phi X174 assay. RNA primase was found to be an ancillary enzyme activity of the DNA polymerase alpha from both carcinogen-treated and untreated cells; however, the RNA primase:DNA polymerase alpha activity ratio was significantly higher in DNA polymerase alpha-primase complexes from carcinogen-treated cells. These complexes also exhibited an at least 3 times greater velocity of synthesis with supercoiled or unprimed single-stranded DNAs as templates. Since the binding sites of DNA polymerase alpha-primase complexes for deoxynucleotide triphosphates and DNA templates were shown to be identical before and after treatment of cells with carcinogens (i.e., identical Km values for different DNA templates and Ki values for specific inhibitors), the increased synthesis catalyzed by the DNA polymerase alpha-primase complex from carcinogen-treated cells might be due to a carcinogen-induced alteration of an accessory protein of the complex.

Animals↗
PubMed1993

Fidelity studies of the human DNA polymerase alpha. The most conserved region among alpha-like DNA polymerases is responsible for metal-induced infidelity in DNA synthesis.

Mutational studies in the highly conserved region I domain of the human DNA polymerase alpha enzyme demonstrated a change in metal cation-specific catalysis. Here, we extend the investigation to include the fidelity of DNA synthesis by these mutants, studying misinsertion, mispair extension, and the nucleotide analog utilization. The fidelity of region I mutants and wild type human DNA polymerase alpha enzyme were analyzed with either Mg2+ or Mn2+ as the metal activator. Despite the known mutagenic effect of Mn2+ in causing polymerases to misinsert nucleotides and to utilize dideoxynucleotides, we have found that two region I mutants, D1002N and T1003S, which utilize Mn2+ in catalysis more effectively than Mg2+, actually have a 70- and 40-fold higher misinsertion fidelity, respectively, in Mn(2+)-catalyzed reactions than that of the wild type enzyme. The enhanced misinsertion fidelity of these two mutants in Mn(2+)-catalyzed reactions is due to Km discrimination of the incorrect nucleotide where the D1002N and T1003S had a 850- and 62-fold higher Km for insertion of incorrect than correct nucleotide, respectively. In Mg(2+)-catalyzed reactions, all of the region I mutants exhibited similar misinsertion efficiencies as the wild type polymerase. Study of mispair extension showed that in Mn(2+)-catalyzed ractions, the wild type polymerase alpha enzyme readily extended mispair termini. In contrast, the two region I mutants, D1002N and T1003S, were unable to extend the mispaired termini in either Mg(2+)- or Mn(2+)-catalyzed reactions. These results suggest that the side chains of region I amino acids play an essential role in the Mn(2+)-induced infidelity during DNA synthesis by human DNA polymerase alpha. The effects of the metal activator on the utilization of two nucleotide analogs, 3'-azido-3'-deoxythymidine triphosphate and ddCTP, by the region I mutants were also investigated.

Base Sequence↗
PubMed2000

In vitro strand transfer from broken RNAs results in mismatch but not frameshift mutations.

An in vitro system to compare the fidelity of strand transfers from truncated vs full-length RNAs was constructed. A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer. All RNAs were derived from the N-terminal portion of the alpha-lac gene. On full-length donors, transfers occurred when DNAs migrated to the acceptor prior to being completed on the donor. On donors that were truncated, most transfers occurred after DNAs reached the end of the donor. Transfer products were amplified by PCR and used to replace the corresponding region in a vector containing the alpha-lac gene. Transformed Escherichia coli were screened for alpha-complementation by blue-white phenotype analysis, with white colonies scored as those with errors in alpha-lac. These errors were derived from RT synthesis and strand transfer. The mutant colony frequency approximately doubled for transfer products derived from truncated donors (0.026+/-0.005 vs. 0.053+/-0.011 (three experiments +/- SD), for full-length vs. truncated, respectively). The increases resulted from additional non-template-directed bases (mostly thymidines) added to the DNAs before transfer. Sequence analysis of DNAs synthesized on truncated donors showed that about 60% had additions (20/34); however, those without additions transferred at a much higher rate than those with. Transfer of the DNAs with additions always resulted in substitutions; no frameshifts were observed. Results are consistent with RT adding nontemplated nucleotides at template termini. Transfer and subsequent extension of these products is severely inhibited relative to products without additions. The potential relevance of these findings to retrovirus replication is discussed.

Base Pair Mismatch↗
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↗
PubMed1994

Misincorporation and mispaired primer extension by human immunodeficiency virus reverse transcriptase.

Pre-steady-state methods were used to study the fidelity of human immunodeficiency virus reverse transcriptase. Fidelity of DNA-directed DNA synthesis can be attributed to a 1-2 order of magnitude reduction in affinity for noncomplementary dNTPs, and a 1-4 order of magnitude reduction in the rate of the conformational change that limits the rate of nucleotide addition. Affinities of reverse transcriptase for paired or mispaired primer termini are similar. Discrimination against a mispaired primer is due to reduction in affinity for the next dNTP and reduction in rate of extension. Extension of mispaired termini proceeds 20-700-fold faster than the rate of dissociation of reverse transcriptase from the primer-template and is 2-3 orders of magnitude more frequent than nucleotide misincorporation. The rate-limiting step for extension of a mispaired terminus occurs at the conformational change or chemical step, depending on the nature of the mispair. Presence of a mismatch at the 3' penultimate position reduces pyrophosphorolysis of the primer by a factor of 10(3), indicating that mispairs 5' to the site of chemistry can also affect catalysis.

Base Composition↗
PubMed2003

The role of Cdh1p in maintaining genomic stability in budding yeast.

Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G(1) transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1delta and mad2delta single mutants, the mad2delta cdh1delta double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2delta cdh1delta and pds1delta cdh1dDelta strains were rescued by overexpressing Swe1p, a G(2)/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1delta mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.

Carrier Proteins↗