Catalytic properties of chloroplast F1-ATPase modified at catalytic or noncatalytic sites by 2-azido adenine nucleotides

When heat-activated F1-ATPase from chloroplasts was repeatedly exposed to Mg2+ and 2-azido-ATP, followed by separation from medium nucleotides and photolysis, a total of two sites per enzyme, both catalytic and noncatalytic, were labeled. In a coupled assay with pyruvate kinase about half the activity was lost when one site per enzyme was modified. However, increased modification resulted in no further loss of activity. In contrast, methanol-sulfite activation of the enzyme showed a loss of most of the catalytic capacity when one site per enzyme was modified. Predominant labeling of either one catalytic or one noncatalytic site caused a loss of most of the activity in either assay. An indication that the enzyme modified at one site retained some catalytic activity was verified by measurement of the [18O]Pi species formed when [gamma-18O]ATP was hydrolyzed by partially derivatized enzyme. With either catalytic or noncatalytic site modification, the distributions of [18O]Pi species formed showed that the modified enzyme had different catalytic characteristics. An interpretation is that with modification by azido nucleotides at either catalytic or noncatalytic sites, capacity for rapid catalysis is largely lost but the remaining sites retain weak modified catalytic properties.     

Deletion analysis in the catalytic region of the 10-23 DNA enzyme

In this study, the functional relevance of the core nucleotides of the RNA cleaving 10-23 DNA enzyme (DNAzyme) was investigated. Systematic deletion studies revealed that DNAzymes lacking thymine at position 8 (T8) retain catalytic activity comparable to that of the wild-type enzyme. Deletion of the adjacent cytosine at position 7 (C7) also resulted in a highly active enzyme and even the double deletion mutant C7/T8 displayed cleavage activity, although the catalytic rate under multiple turnover conditions was found to be reduced by one order of magnitude. The identification of non-essential nucleotides in the catalytic core might help to stabilize the DNAzyme against nucleolytic degradation and to overcome problems in elucidating its three-dimensional structure.     

Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI

The cleavage of specific DNA sequences by the restriction endonucleases AluI, DdeI, HinfI, RsaI, and TaqI has been studied by monitoring the effect of various nucleotide modifications on the rate of DNA digestion. Bacteriophage fd DNA was completely substituted in one strand with a single nucleotide analog, using an in vitro primed DNA synthesis reaction on a single-stranded viral DNA template. Twelve deoxynucleotide analogs were incorporated into these DNA substrates: 2-aminopurine, 2,6-diaminopurine, deoxytubercidin, deoxyuridine, 5-bromodeoxyuridine, 5-allylamine deoxyuridine, 5-biotinyl deoxyuridine, deoxypseudouridine, deoxyinosine, 8-azadeoxyguanosine, 5-iododeoxycytidine, and 5-bromodeoxycytidine. The restriction enzymes tested varied considerably in their ability to digest hemi-substituted DNAs containing these modified nucleotides. Structural alterations in the base pairs immediately adjacent to the phosphodiester bonds cleaved by the enzyme reduced the rate of enzyme activity most dramatically, and in most cases more than a single determinant on each base pair altered activity. Interactions with nucleotides outside the recognition site seem to have little importance in the binding or catalytic activity of these enzymes.     

Direct involvement of the TEN domain at the active site of human telomerase

Telomerase is a ribonucleoprotein that adds DNA to the ends of chromosomes. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is important for activity and processivity. Here we describe a mutation in the TEN domain of human TERT that results in a greatly increased primer K(d), supporting a role for the TEN domain in DNA affinity. Measurement of enzyme kinetic parameters has revealed that this mutant enzyme is also defective in dNTP polymerization, particularly while copying position 51 of the RNA template. The catalytic defect is independent of the presence of binding interactions at the 5'-region of the DNA primer, and is not a defect in translocation rate. These data suggest that the TEN domain is involved in conformational changes required to position the 3'-end of the primer in the active site during nucleotide addition, a function which is distinct from the role of the TEN domain in providing DNA binding affinity.     

Factors Affecting the Tailing of Blunt End DNA with Fluorescent Pyrimidine dNTPs

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3′ ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson–Crick base pair at the end. Mismatched duplexes with a 3′ dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5′ exonuclease activity of the enzyme.     

Changes in DNA and RNA synthesis and associated enzyme activities after the stimulation of serum-depleted BHK21-C13 cells by the addition of serum

BHK21/C13 cells placed in medium containing low (1%) serum ceased DNA synthesis within 4 days. DNA synthesis recommenced 10 h after the readdition of serum (to 10%) to cells incubated for 6 days in serum-depleted medium. Two peaks of thymidine incorporation were observed at 12–13 h and 15–17 h, followed by a single peak of dividing cells at 25 h. The two peaks of incorporation represent variation in the extent of DNA replication during a single synchronous S phase.Uridine, deoxyadenosine and deoxyguanosine kinase activities did not decline in serum-depleted cells and, after the addition of serum, their activities showed cyclical variation about a mean involving two-fold changes in enzyme specific activity. All other enzyme activities examined were markedly decreased in resting cells.Ornithine decarboxylase activity increased 15-fold within 5 h of serum addition, but had returned to the resting level by 8 h. There was no apparent correlation between this alteration of enzyme activity and the rate of RNA synthesis.DNA polymerase, thymidine kinase and deoxycytidine kinase activities all decreased further within 4 h of the addition of serum, followed by several-fold increases in activity. The peak of DNA polymerase activity corresponded to, and encompassed, both peaks of DNA synthesis. However, thymidine and deoxycytidine kinase activities, although exhibiting two activity maxima corresponding to the peaks of DNA synthesis, were at their highest levels in G2.     

Modified binary hammerhead ribozymes with high catalytic activity

A series of binary hammerhead ribozymes was designed and assessed in terms of cleavage activity and nuclease resistance. Enhanced nuclease resistance of binary ribozymes was achieved by incorporation of Z-modified nucleotides at the selective positions along with addition of 3'-3-linked thymidine cap. These modified binary ribozymes efficiently cleave 190-nucleotides long MDR1 mRNA fragment and display catalytic activity much higher then respective full-length analogs.     

The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I

The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.     

MODIFIED BINARY HAMMERHEAD RIBOZYMES WITH HIGH CATALYTIC ACTIVITY

A series of binary hammerhead ribozymes was designed and assessed in terms of cleavage activity and nuclease resistance. Enhanced nuclease resistance of binary ribozymes was achieved by incorporation of 2′-modified nucleotides at the selective positions along with addition of 3′-3′-linked thymidine cap. These modified binary ribozymes efficiently cleave 190-nucleotides long MDR1 mRNA fragment and display catalytic activity much higher then respective full-length analogs.     

Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii

New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage, suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of uridine 5′-monophosphate (UMP) and thymidine 5′-monophosphate (dTMP) pools, thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CPSII) induces a severe uracil auxotrophy with no detectable parasite replication in vitro and complete attenuation of virulence in mice. Here we show that a CPSII cDNA minigene efficiently complements the uracil auxotrophy of CPSII-deficient mutants, restoring parasite growth and virulence. Our complementation assays reveal that engineered mutations within, or proximal to, the catalytic triad of the N-terminal glutamine amidotransferase (GATase) domain inactivate the complementation activity of T. gondii CPSII and demonstrate a critical dependence on the apicomplexan CPSII GATase domain in vivo. Surprisingly, indels present within the T. gondii CPSII GATase domain as well as the C-terminal allosteric regulatory domain are found to be essential. In addition, several mutations directed at residues implicated in allosteric regulation in Escherichia coli CPS either abolish or markedly suppress complementation and further define the functional importance of the allosteric regulatory region. Collectively, these findings identify novel features of T. gondii CPSII as potential parasite-selective targets for drug development.     

The structure-function relationship in the clostripain family of peptidases.

In this study we investigate the active-site structure and the catalytic mechanism of clostripain by using a combination of three separate techniques: affinity labelling, site-directed mutagenesis and molecular modelling. A benzamidinyl-diazo dichlorotriazine dye (BDD) was shown to act as an efficient active site-directed affinity label for Clostridium histolyticum clostripain. The enzyme, upon incubation with BDD in 0.1 m Hepes/NaOH buffer pH 7.6, exhibits a time-dependent loss of activity. The rate of inactivation exhibits a nonlinear dependence on the BDD concentration, which can be described by reversible binding of dye to the enzyme prior to the irreversible reaction. The dissociation constant of the reversible formation of an enzyme-BDD complex is KD = 74.6 +/- 2.1 micro m and the maximal rate constant of inactivation is k3 = 0.21 x min(-1). Effective protection against inactivation by BDD is provided by the substrate N-benzoyl-L-arginine ethyl ester (BAEE). Cleavage of BDD-modified enzyme with trypsin and subsequent separation of peptides by reverse-phase HPLC gave only one modified peptide. Amino acid sequencing of the modified tryptic peptide revealed the target site of BDD reaction to be His176. Site-directed mutagenesis was used to study further the functional role of His176. The mutant His176Ala enzyme exhibited zero activity against BAEE. Together with previous data, these results confirm that a catalytic dyad of His176 and Cys231 is responsible for cysteine peptidase activity in the C11 peptidase family. A molecular model of the catalytic domain of clostripain was constructed using a manually extended fold recognition-derived alignment with caspases. A rigorous iterative modelling scheme resulted in an objectively sound model which points to Asp229 as responsible for defining the strong substrate specificity for Arg at the P1 position. Two possible binding sites for the calcium required for auto-activation could be located. Database searches show that clostripain homologues are not confined to bacterial lineages and reveal an intriguing variety of domain architectures.     

The linker region between the helicase and primase domains of the gene 4 protein of bacteriophage T7. Role in helicase conformation and activity

The gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for template-directed oligoribonucleotide synthesis. A 26 amino acid linker region (residues 246-271) connects the two domains and is essential for the formation of functional hexamers. In order to further dissect the role of the linker region, three residues (Ala257, Pro259, and Asp263) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 phage lacking gene 4. The in vivo screening revealed Ala257 and Asp263 to be essential whereas Pro259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala257 or Asp263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg2+, all of the altered proteins oligomerize. However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation. Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA. Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required. The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer.     

Mitochondrial F1-ATPase will bind and cleave ATP but only slowly release ADP after N,N'-dicyclohexylcarbodiimide or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatization

The ATPase from the inner mitochondrial membrane is known to be inhibited by modification of one of the three catalytic subunits with N,N'-dicyclohexylcarbodiimide (DCCD) or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. An experimental approach described in this paper shows that most of the residual ATPase activity observed after the usual DCCD or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole modification is due to the presence of unmodified enzyme, although the large fraction of modified enzyme retains a weak catalytic activity. This weak catalytic activity can be stimulated by methanol or dimethyl sulfoxide. When the modified enzymes are exposed to Mg2+ and [3H]ATP, about equal amounts of [3H]ATP and [3H]ADP appear at catalytic sites. The turnover rate for these enzymes is less than 1/1000 that of the native enzyme when it is calculated from the rate at which the enzyme becomes labeled at the catalytic sites with [3H]ATP and [3H]ADP during steady state hydrolysis. In addition, a higher ATP concentration is required for steady state turnover and, after ATP binding, the principal rate-limiting step is the capacity of the derivatized enzyme to undergo the binding changes necessary for the release of ADP and Pi. When the modified enzymes are not hydrolyzing ATP, they convert to form(s) that show a distinct lag in the replacement of bound nucleotides at catalytic sites. The replacement of bound nucleotides is still promoted by MgATP, even though the enzymes have been converted to sluggish forms. Contrary to a recent suggestion based on the study of the DCCD-modified enzyme (Soong, K.S., and Wang, J.H. (1984) Biochemistry 23, 136-141), our data provide evidence for the existence of catalytic cooperatively between at least two alternating sites in the modified enzyme and are consistent with continued sequential participation of all three sites.     

Identification of two new nucleotide mutations (HPRTUtrecht and HPRTMadrid) in exon 3 of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene

Mutations in the X-linked hypoxanthine-guanine phosphoribosyl transferase gene (HPRT) result in deficiencies of HPRT enzyme activity, which may cause either a severe form of gout or Lesch-Nyhan syndrome depending on the residual enzyme activity. Mutations leading to these diseases are heterogeneous and include DNA base substitutions, DNA deletions, DNA base insertions and errors in RNA splicing. Identification of mutations has been performed at the RNA and DNA level. Sequencing genomic DNA of the HPRT gene offers the possibility of direct diagnostic analysis independent on the expression of the mature HPRT mRNA. We describe a Dutch and a Spanish family, in which the Lesch-Nyhan syndrome and a severe partial HPRT-deficient phenotype, respectively, were diagnosed. Direct sequencing of the exons coding for the HPRT gene was performed in both families. Two new exon 3 mutations have been identified. At position 16676, the normally present G was substituted by an A in the Dutch kindred (HPRT_Utrecht), and led to an arginine for glycine change at residue 70. At position 16680, the G was substituted by a T in the Spanish family (HPRT_Madrid); this substitutes a valine for glycine at residue 71. These new mutations are located within one of the clusters of hotspots in exon 3 of the HPRT gene in which HPRT_Yale and HPRT_{New Haven} have previously been identified.     

Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain.

Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.     

Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

Poly(3-hydroxybutyrate) (PHB) depolymerase from Alcaligenes faecalis T1 is composed of three domains: the catalytic (C) domain, the fibronectin type III-like (F) domain, and the substrate-binding (S) domain. We constructed domain deletion, inversion, chimera, and extra-F-domain mutants and examined their enzyme activity and PHB-binding ability. In addition, we performed substitution of 214Asp and 273His with glycine and aspartate, respectively, to examine their participation in a catalytic triad together with 139Ser. The mutant with both the F and S domains deleted and the trypsin-digested enzyme showed no PHB-hydrolyzing activity and less PHB-binding ability than that of the wild-type enzyme but retained D-(-)-3-hydroxybutyrate trimer-hydrolyzing activity at a level similar to that of the wild-type enzyme. The mutant with the F domain deleted and the mutant which had the order of the F and S domains inverted retained PHB-binding ability and trimer-hydrolyzing activity at levels similar to those of the wild-type enzyme but lost PHB-hydrolyzing activity. The chimera mutant, in which the F domain was substituted with a Thr-rich domain of PHB depolymerase A from Pseudomonas lemoignei, and the extra-F-domain mutant, with an additional F domain, retained trimer- and PHB-hydrolyzing activities and PHB-binding ability at levels similar to those of the wild-type enzyme. Two mutants (D214G and H273D) showed no enzymatic activity toward trimer and PHB, and they were not labeled with [3H]diisopropylfluorophosphate.     

Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride

Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.     

Inosine 5'-monophosphate vs inosine and hypoxanthine as substrates for purine salvage in human lymphoid cells

The ability of inosine 5'-monophosphate vs inosine or hypoxanthine to supply the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells was evaluated. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and make the cells dependent upon an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 25 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA at rates equal to that of untreated control cultures. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line, WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line No. 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, only inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells and suggest that this enzyme may have functional significance when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.     

Structure and function of the mouse DNA methyltransferase gene: Dnmt1 shows a tripartite structure

Dnmt1 is the predominant DNA methyltransferase (MTase) in mammals. The C-terminal domain of Dnmt1 clearly shares sequence similarity with many prokaryotic 5mC methyltransferases, and had been proposed to be sufficient for catalytic activity. We show here by deletion analysis that the C-terminal domain alone is not sufficient for methylating activity, but that a large part of the N-terminal domain is required in addition. Since this complex structure of Dnmt1 raises issues about its evolutionary origin, we have compared several eukaryotic MTases and have determined the genomic organization of the mouse Dnmt1 gene. The 5' most part of the N-terminal domain is dispensible for enzyme activity, includes the major nuclear import signal and comprises tissue-specific exons. Interestingly, the functional subdivision of Dnmt1 correlates well with the structure of the Dnmt1 gene in terms of intron/exon size distribution as well as sequence conservation. Our results, based on functional, structural and sequence comparison data, suggest that the gene has evolved from the fusion of at least three genes.     

Identification and properties of the catalytic domain of mammalian DNA polymerase beta

Rat DNA polymerase beta (beta-pol) is a 39-kDa protein organized in two tightly folded domains, 8-kDa N-terminal and 31-kDa C-terminal domains, connected by a short protease-sensitive region. The 8-kDa domain contributes template binding to the intact protein, and we now report that the 31-kDa C-terminal domain contributes catalytic activity. Our results show that this domain as a purified proteolytic fragment conducts DNA synthesis under appropriate conditions but the kcat is lower and primer extension properties are different from those of the intact enzyme. A proteolytic truncation of the 31-kDa catalytic domain fragment, to remove a 60-residue segment from the NH2-terminal end, results in nearly complete loss of activity, suggesting the importance of this segment. Overall, these results indicate that the domains of beta-pol have distinct functional roles, template binding and nucleotidyltransferase, respectively; yet, the intact protein is more active for each function than the isolated individual domain fragment.