Active-site alkylation destabilizes human O6-alkylguanine DNA alkyltransferase

O(6)-alkylguanine-DNA alkyltransferase (AGT) repairs pro-mutagenic O(6)-alkylguanine and O(4)-alkylthymine lesions in DNA. The alkylated form of the protein is not reactivated; instead, it is rapidly ubiquitinated and degraded. Here, we show that alkylation destabilizes the native fold of the protein by 0.5-1.2 kcal/mole and the DNA-binding function by 0.8-1.4 kcal/mole. On this basis, we propose that destabilization of the native conformational ensemble acts as a signal for ubiquitination.     

Increased resistance to the toxic effects of alkylating agents in tobacco expressing the E. coli DNA repair gene ada.

The protein coding region of the E. coli gene ada has been transferred to tobacco plants by a leaf disc transformation procedure involving an Agrobacterium tumefaciens Ti plasmid. Transformed plants were shown to be transgenic for the ada message and had increased levels of O6-alkylguanine DNA alkyltransferase activity. The N-methyl-N-nitrosourea- or taurinechlorethylnitrosourea-induced inhibition of growth of calluses or of cells in suspension was considerably lower in ada-transformed than in non-transformed plants. This indicates that O6-alkylguanine, O4-alkylthymine or phosphotriesters are growth-inhibitory lesions in tobacco.     

Sequence-dependent flexibility in promoter sequences

The non-neighbor interactions between base-pairs were taken into account to calculate the angular parameters (Omega, rho and tau) describing the orientation of successive base-pair planes and the translation parameters (D(y)) along the long axis of base-pair steps for 36 independent tetramers. A statistical mechanical model was proposed to predict the DNA flexibility that is mainly related to the thermal fluctuations at individual base-pair steps. The DNA flexibility can be described by the root-mean-square deviation of the end-to-end distance of DNA helical structure. The present model was then used to investigate the extreme flexible pattern in prokaryotic and eukaryotic promoter sequences. The results demonstrated several extreme flexible regions related to functionally important elements exist both in prokaryotic promoters and in eukaryotic promoters, DNA flexibility and AT content are highly correlated. The probabilities finding flexibility pattern in promoter sequences were also estimated statistically. The biological implications were discussed briefly.     

A simple method for the solid phase synthesis of oligodeoxynucleotides containing O4-alkylthymine.

A route to prepare the cyanoethyl-phosphoramidite monomer of O4-alkylthymine and a method for the routine solid-phase synthesis of oligodeoxynucleotides containing O4-alkylthymine are described. This method has been used to make DNA sequences up to 48 bases in length. The amino function of the adenine and guanine in the sequence were protected with the phenoxyacetyl group, and that of cytosine with the isobutyryl group. The phosphodiesters were protected with the cyanoethyl group. This allowed complete deprotection of the oligomer with alkoxide ions (methanol/1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) for the oligomers containing O4-methylthymine, or ethanol/DBU for those containing O4-ethylthymine) thus avoiding the use of ammonia which is known to attack the O4-alkylthymine to form 5-methylcytosine. There was no detectable loss of the alkyl group to form thymine.     

Solution structure of a DNA double helix incorporating four consecutive non-Watson-Crick base-pairs

A series of DNA 21-mers containing a variety of the 4 x 4 internal loop sequence 5'-CAAG-3'/3'-ACGT-5' were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers exhibit excellent resolution in the NMR spectra and reveal many unusual NOEs (nuclear Overhauser effect) that allow for the detailed characterization of a DNA hairpin incorporating a track of four different non-Watson-Crick base-pairs in the stem. These include a wobble C.A base-pair, a sheared A.C base-pair, a sheared A.G base-pair, and a wobble G.T base-pair. Significantly different twisting angles were observed between the base-pairs in internal loop that results with excellent intra-strand and inter-strand base stacking within the four consecutive mismatches and the surrounding canonical base-pairs. This explains why it melts at 52 degrees C even though five out of ten base-pairs in the stem adopt non-Watson-Crick pairs. However, the 4 x 4 internal loop still fits into a B-DNA double helix very well without significant change in the backbone torsion angles; only zeta torsion angles between the tandem sheared base-pairs are changed to a great extent from the gauche(-) domain to the trans domain to accommodate the cross-strand base stacking in the internal loop. The observation that several consecutive non-canonical base-pairs can stably co-exist with Watson-Crick base-pairs greatly increases the limited repertoire of irregular DNA folds and reveals the possibility for unusual structural formation in the functionally important genomic regions that have potential to become single-stranded.     

rII cistrons of bacteriophage T4. DNA sequence around the intercistronic divide and positions of genetic landmarks

An 873 base-pair DNA sequence from the rII region of bacteriophage T4 is presented. The sequence encodes 139 carboxyl-terminal amino acids of rIIA and the amino-terminal 146 amino acids of rIIB. Eleven base-pairs separate the rIIA stop codon (UAA) and the rIIB AUG.An extensive genetic map is superimposed on the DNA sequence, showing the deduced locations of many of the mutations (base-pair substitutions, frameshifts, deletions) found in previous rII genetic studies.     

Partial suppression of an ochre mutation in Saccharomyces cerevisiae by multicopy plasmids containing a normal yeast tRNAGln gene

We screened a yeast genomic library for recombinant DNA plasmids that complemented the ultraviolet (u.v.) sensitivity of a strain of Saccharomyces cerevisiae designated rad4-3 that is defective in excision repair of DNA. A multicopy plasmid (pNF4000) with a 9.4 X 10(3) base-pair yeast DNA insert partially complemented the u.v. sensitivity of rad4-3, but not of two other rad4 allelic mutants (rad4-2 and rad4-4), or of other u.v.-sensitive rad mutants. The yeast insert was analyzed by restriction mapping, DNA-DNA hybridization, DNA-tRNA hybridization and DNA sequencing. This analysis revealed the presence of a normal tRNAGln gene, a yeast sigma element situated 5' to the transfer RNA gene, a Ty element and a solo delta element. Deletion analysis of pNF4000 showed that the tRNAGln gene is required for partial complementation of the u.v. sensitivity of rad4-3. Furthermore, a multicopy plasmid containing a tRNAGln gene derived from a different region of the yeast genome also partially complemented the u.v. sensitivity of rad4-3. The rad4-3 mutation is suppressed following transformation with a plasmid containing the known ochre suppressor SUP11-o, indicating that it is an ochre mutation. We therefore conclude that when expressed in sufficient quantity, normal tRNAGln (which usually decodes the sense codon CAA) can weakly suppress the nonsense ochre codon UAA, and suggest that this represents an example of wobble occurring at the first rather than at the third position of the codon.     

Bent DNA is needed for recombinational enhancer activity in the site-specific recombination system Cin of bacteriophage P1. The role of FIS protein

A series of recombinational enhancer mutants was constructed by manipulating the ClaI site between the two FIS binding sites of the Hin enhancer. These mutants include insertions from two to 12 base-pairs and two deletions of one or two base-pairs. Recombinational enhancer activity was found only with four mutants carrying either a four base-pair substitution, ten base-pair insertions or a one base-pair deletion, respectively; two other ten base-pair insertion mutants, however, were inactive, although FIS protein binding was unaffected. So, besides binding of FIS protein to its specific sites within the enhancer sequence and the correct helical positioning of these sites on the DNA, another criterion for enhancer activity must be fulfilled. DNA bending assays identify this requirement as a change of the enhancer DNA conformation, which FIS protein is able to induce and to stabilize. This conformational change of the DNA can be blocked by mutations in the central segment between the two FIS binding sites of the Hin enhancer. This sequence has special functions for the recombinational enhancer activity.     

Interactions of human O6-alkylguanine-DNA alkyltransferase (AGT) with short single-stranded DNAs

The O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. Here we characterize the binding of AGT to single-stranded DNAs ranging in length from 5 to 78 nucleotides (nt). Binding is moderately cooperative (37.9 +/- 3.0 相似文献   

Methyl transferases induced during chemical adaptation of M. luteus.

Three peaks of methyltransferase activity specific for MNNG alkylated DNA have been identified from extracts of chemically adapted M. luteus. They are designated as TI to TIII in order to their elution from a Sephadex G-75 column. The first one of these peaks has been purified to homogeneity. TI, is an inducible, unusually salt resistant, heat labile protein which corrects O6-methylguanine in alkylated DNA by the transfer of the O6-alkyl group to a cysteine amino acid in the TI protein. There is a stoichiometric relationship between the loss of O6-methylguanine from the DNA and the production of S-methylcysteine. Partially purified TII & TIII proteins show specificity for O4-alkylthymine and methyl phosphotriesters respectively. The mode of repair by the isolated methyltransferases is similar yet there is no competition for substrate specificity. The apparent molecular weights of TI, TII & TIII proteins are 31Kd, 22Kd, and 13Kd respectively.     

Interactions of DNA binding ligands with PNA-DNA hybrids.

The interactions of two representative mixed-sequence (one with an AT-stretch) PNA-DNA duplexes (10 or 15 base-pairs) and a PNA2/DNA triplex with the DNA binding reagents distamycin A, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, 8-methoxy-psoralen and the delta and lambda enantiomers of Ru(phen)2-dppz2+ have been investigated using optical spectroscopic methods. The behaviour of these reagents versus two PNA-PNA duplexes has also been investigated. With triple helical poly(dA)/(H-T10-Lys-NH2)2 no significant intercalative binding was detected for any of the DNA intercalators, whereas DAPI, a DNA minor groove binder, was found to exhibit a circular dichroism with a positive sign and amplitude consistent with minor groove binding. Similarly, a PNA-DNA duplex containing a central AATA motif, a typical minor groove binding site for the DNA minor groove binders distamycin A and DAPI, showed binding for both of these drugs, though with strongly reduced affinity. No important interactions were found for any of the ligands with a PNA-DNA duplex consisting of a ten base-pair mixed purine-pyrimidine sequence with only two AT base-pairs in the centre. Nor did any of the ligands show any detectable binding to the PNA-PNA duplexes (one containing an AATT motif). Various PNA derivatives with extentions of the backbone, believed to increase the flexibility of the duplex to opening of an intercalation slot, were tested for intercalation of ethidium bromide or 8-methoxypsoralen into the mixed sequence PNA-DNA duplex, however, without any observation of improved binding. The importance of the ionic contribution of the deoxyribose phosphate backbone, versus interactions with the nucleobases, for drug binding to DNA is discussed in the light of these findings.     

Mutations that probe the cooperative assembly of O⁶-alkylguanine-DNA alkyltransferase complexes

O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O(6)-alkylguanine and O(4)-alkylthymine adducts present in DNA that has been exposed to alkylating agents. AGT binds DNA cooperatively, and models of cooperative complexes predict that residues 1-7 of one protein molecule and residues 163-169 of a neighboring protein are closely juxtaposed. To test these models, we used directed mutagenesis to substitute triplets of alanine for triplets of native residues across these two sequences. Six of eight designed mutants expressed AGT at detectable levels. All mutant AGTs that were expressed were folded compactly, bound DNA with stoichiometries equivalent to that of the wild-type protein, and were able to protect Escherichia coli to varying degrees from the potent alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). All mutations attenuated DNA binding cooperativity, but unexpectedly, they also reduced the affinity of AGT for DNA. This suggests that the protein-protein and protein-DNA interactions of AGT are strongly coupled. When normalized for differences in AGT expression, cells expressing mutants KDC(3-5)-AAA, DCE(4-6)-AAA, and KEW(165-167)-AAA were significantly more susceptible to MNNG than wild-type cells. This is the first evidence, to the best of our knowledge, of a role for residues at the protein-protein interface and, by implication, cooperative protein-protein interactions in the cell-protective mechanisms of AGT.     

Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA

The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila.     

Chromatin assembly on plasmid DNA in vitro. Apparent spreading of nucleosome alignment from one region of pBR327 by histone H5.

We have found that histone H5 (or H1) induces physiological nucleosome spacings and extensive ordering on some plasmid constructions, but not on others, in a fully defined in vitro system. Plasmid pBR327 containing DNA insertions with lengths close to 300 base-pairs permitted histone H5 to induce a remarkable degree of nucleosome alignment. Seventeen multiples of a unit 210(+/- 4) base-pair repeat, covering the entire plasmid, were detected. Plasmid pBR327, not containing a DNA insert, permitted continuous alignment of only a few nucleosomes. These observations suggest that a necessary requirement in this system for histone H5 (or H1)-induced nucleosome alignment on small (less than 4 kb; 1 kb = 10(3) bases or base-pairs) circular plasmids may be that the total DNA length must be close to an integer multiple of the nucleosome repeat length generated, a type of boundary effect. Consistent with this hypothesis, five deletion constructs of pBR327 (not containing inserts), that spanned 64% of the plasmid, and possessed DNA lengths close to integer multiples of 210 base-pairs, permitted nucleosome alignment by histone H5. We have also found that plasmid length adjustment is not a sufficient condition for nucleosome alignment. For example, plasmids pBR322 and pUC18 did not permit nucleosome alignment when adjusted to near-integer multiples of 210 base-pairs. Also, for pBR327 that contained a length-adjusted deletion in one particular region, appreciable nucleosome alignment no longer occurred. These data suggest that a contiguous approximately 800 base-pair region of pBR327, interrupted in pBR322 and not present in pUC18, can nucleate histone H5-induced nucleosome alignment, which can then spread to adjacent chromatin. Supporting this idea, a positioned five-nucleosome array appears to originate in the required region. Additionally, on a larger (6.9 kb) plasmid construction, the "chromatin organizing region" of pBR327 and adjacent DNA on one side of it exhibited preferred H5-induced nucleosome alignment.     

Solution structure of an 11-mer duplex containing the 3, N(4)-ethenocytosine adduct opposite 2'-deoxycytidine: implications for the recognition of exocyclic lesions by DNA glycosylases

Lipid peroxidation products, as well as the metabolic products of vinyl chloride, react with cellular DNA producing the mutagenic adduct 3,N(4)-etheno-2'-deoxycytidine (epsilondC), along with several other exocyclic derivatives. High-resolution NMR spectroscopy and restrained molecular dynamics simulations were used to establish the solution structure of an 11-mer duplex containing an epsilondC.dC base-pair at its center. The NMR data suggested a regular right-handed helical structure having all residues in the anti orientation around the glycosydic torsion angle and Watson-Crick alignments for all canonical base-pairs of the duplex. Restrained molecular dynamics generated a three-dimensional model in excellent agreement with the spectroscopic data. The (epsilondC. dC)-duplex structure is a regular right-handed helix with a slight bend at the lesion site and no severe distortions of the sugar-phosphate backbone. The epsilondC adduct and its partner dC were displaced towards opposite grooves of the helix, resulting in a lesion-containing base-pair that was highly sheared but stabilized to some degree by the formation of a single hydrogen bond. Such a sheared base-pair alignment at the lesion site was previously observed for epsilondC.dG and epsilondC.T duplexes, and was also present in the crystal structures of duplexes containing dG.T and dG. U mismatches. These observations suggest the existence of a substrate structural motif that may be recognized by specific DNA glycosylases during the process of base excision repair.     

Base sequence and helix structure variation in B and A DNA

The observed propeller twist in base-pairs of crystalline double-helical DNA oligomers improves the stacking overlap along each individual helix strand. But, as proposed by Calladine, it also leads to clash or steric hindrance between purines at adjacent base-pairs on opposite strands of the helix. This clash can be relieved by: (1) decreasing the local helix twist angle between base-pairs; (2) opening up the roll angle between base-pairs on the side on which the clash occurs; (3) separating purines by sliding base-pairs along their long axes so that the purines are partially pulled out of the stack (leading to equal but opposite alterations in main-chain torsion angle delta at the two ends of the base-pair); and (4) flattening the propeller twist of the offending base-pairs. Simple sum functions, sigma 1 through sigma 4, are defined, by which the expected local variation in helix twist, base roll angle, torsion angle delta and propeller twist may be calculated from base sequence. All four functions are quite successful in predicting the behavior of B DNA. Only the helix twist and base roll functions are applicable to A DNA, and the helix twist function begins to fail for an A helical RNA/DNA hybrid. Within these limits, the sequence-derived sum functions match the observed helix parameter variation quite closely, with correlation coefficients greater than 0.900 in nearly all cases. Implications of this sequence-derived helix parameter variation for repressor-operator interactions are considered.     

Structure-related properties of the mutagenic lesion 6-O-methylguanine in DNA.

Chemical probing of the structures of a few very similar 30 base-pair duplexes containing a 6-O-methylguanine (meG) residue at the 16th position reveals that the modified base simultaneously perturbs the helical structure in two ways; it preferentially unstacks the 3' neighbouring base residue (thymine in this study) on the same strand and it unstacks the pyrimidine to which it is base-paired. Depending on its neighbouring 5' base residue and the base-pairing pyrimidine, this perturbation can extend to a few base-pairs in both 3' and 5' directions from the abnormal base-pair. These perturbations can be detected by cleavage at the site for the restriction enzyme MaeII. The unstaking of the C in the meG.C and A.C base-pairs may explain the de novo methylation of these helices by the human DNA-(cytosine-5-)methyltransferase. Interestingly, the kinetics of repair of the 6-O-methylguanine-containing dinucleotides by the cloned human methylguanine methyltransferase appears to be largely determined by the strength of the stacking interaction between the 6-O-methylguanine and the 5' neighbouring base.     

Molecular mechanics model of supercoiled DNA

We describe a pseudo-atomic model of supercoiled DNA. Each base-pair of the DNA is represented in the model by three particles placed in a plane. The particle triplets are stacked to model stacked base-pairs in double-helical DNA, and closed circular conformations are generated to investigate supercoiling. This model is less detailed than all-atom models, which are too computationally demanding to be used to study supercoiling. On the other hand, this model contains details at the base-pair level and is therefore more elaborate than elastomechanical models. A potential energy function is written in terms of a set of internal co-ordinates defined to resemble a limited number of helical parameters. The modeled helical parameters, helical twist, base-roll, tilt and rise, are the most important parameters of the global shape of DNA. Experimentally measured mechanical properties of DNA are used to define the forces holding the particles together. We then use a procedure incorporating energy minimization and molecular dynamics to locate low energy conformations of the model DNA. The model was found to behave very much like rubber-tubing and elastomechanical models. The conformations and the effects of supercoiling pressure (a number proportional to the degree to which the total twist of the DNA has been altered from its natural value) on these conformations are all very similar to those observed in the latter two models. We also used this model to examine the effects of supercoiling pressure, base-sequence and mechanical properties on the conformations and energies of five sequences. The sequences studied include models of naturally straight DNA and DNA with static or natural bends.     

Effects of DNA sequence and histone-histone interactions on nucleosome placement

Using competitive reconstitution, we have refined the parameters for the binding of histone octamers to artificial nucleosome-positioning sequences of the form: (A/T3nn(G/C)3nn. We find that the optimal period between flexible segments is approximately 10.1 base-pairs, supporting the view that the DNA on the nucleosome surface is overwound. The strongest requirement for flexible DNA is near the protein dyad. However, we see no indication of changes in DNA helical repeat in this region. Using a series of repetitive sequences, we confirm that neither all A/T-rich nor all G/C-rich regions are identical in promoting nucleosome formation. Surprisingly, A/T-rich segments containing the TpA step, subject to purine-purine clash in the minor groove, favor nucleosome formation over sequences lacking this step. Short tracts of adenine residues are found to position on the histone surface like other A/T-rich regions, in the manner predicted by the direction of their sequence-directed bends as determined by electrophoretic methods. Tracts containing five adenine residues are extremely aniostropic in their flexibility and are strongly detrimental to nucleosome formation when positioned for major groove compression. Longer adenine tracts are found to position near the ends of the nucleosomal DNA. However, other positions may be occupied by an A12 tract, with only a minor penalty in the free energy of nucleosome formation. Overall, reconstituted nucleosome positions are translationally degenerate, suggesting a weak dependence on DNA flexibility for nucleosome positioning. Dinucleosomal reconstitutions on tandem dimers of the 5 S RNA gene of Lytechinus variegatus demonstrate a weak phasing dependence for the interaction between nucleosomes. This interaction is maximal for the 202 base-pair repeat and suggests a co-operative mechanism for the formation of ordered nucleosomal arrays based on a combination of DNA flexibility and nucleosome-nucleosome interactions.