ABSTRACT
DNA and RNA are the only known natural genetic materials. Systematic modification of each of their chemical building blocks (nucleobase, sugar, and phosphate) has enabled the study of the key properties that make those nucleic acids genetic materials. All three moieties contribute to replication and, significantly, all three moieties can be replaced by synthetic analogs without loss of function. Synthetic nucleic acid polymers capable of storing and propagating information not only expand the central dogma, but also highlight that DNA and RNA are not unique chemical solutions for genetic information storage. By considering replication as a question of information transfer, we propose that any polymer that can be replicated could serve as a genetic material.
Subject(s)
Carbohydrates/chemical synthesis , DNA/chemistry , Polymers/chemical synthesis , Purines/chemistry , Pyrimidines/chemistry , RNA/chemistry , Base Pairing , Carbohydrates/genetics , DNA/genetics , DNA Replication , Evolution, Molecular , Information Theory , Oligonucleotides/chemistry , Oligonucleotides/genetics , RNA/genetics , ThermodynamicsABSTRACT
We report the crystal structure of release factor 2 bound to ribosome with an aminoacyl tRNA substrate analog at the ribosomal P site, at 3.1 A resolution. The structure shows that upon stop-codon recognition, the universally conserved GGQ motif packs tightly into the peptidyl transferase center. Nucleotide A2602 of 23S rRNA, implicated in peptide release, packs with the GGQ motif in release factor 2. The ribose of A76 of the peptidyl-tRNA adopts the C2'-endo conformation, and the 2' hydroxyl of A76 is within hydrogen-bond distance of the 2' hydroxyl of A2451. The structure suggests how a catalytic water can be coordinated in the peptidyl transferase center and, together with previous biochemical and computational data, suggests a model for how the ester bond between the peptidyl tRNA and the nascent peptide is hydrolyzed.
Subject(s)
Biocatalysis , Peptide Termination Factors/chemistry , Peptide Termination Factors/metabolism , Peptides/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Thermus thermophilus/metabolism , Amino Acid Motifs , Glutamine/metabolism , Hydroxyl Radical/metabolism , Models, Biological , Models, Molecular , Nucleotides/metabolism , Peptide Chain Termination, Translational , Peptidyl Transferases/metabolism , RNA, Transfer, Amino Acyl/metabolism , Substrate Specificity , Water/metabolismABSTRACT
Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially single-stranded DNA with high affinity. They are involved in DNA metabolism in all organisms and serve a vital role in replication, recombination and repair of DNA. In this report, we identify human mitochondrial SSB (HmtSSB) as a novel protein-binding partner of tumour suppressor p53, in mitochondria. It binds to the transactivation domain (residues 1-61) of p53 via an extended binding interface, with dissociation constant of 12.7 (+/- 0.7) microM. Unlike most binding partners reported to date, HmtSSB interacts with both TAD1 (residues 1-40) and TAD2 (residues 41-61) subdomains of p53. HmtSSB enhances intrinsic 3'-5' exonuclease activity of p53, particularly in hydrolysing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) present at 3'-end of DNA. Taken together, our data suggest that p53 is involved in DNA repair within mitochondria during oxidative stress. In addition, we characterize HmtSSB binding to ssDNA and p53 N-terminal domain using various biophysical measurements and we propose binding models for both.
Subject(s)
DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Mitochondrial Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , 8-Hydroxy-2'-Deoxyguanosine , Binding Sites , Cell Line, Tumor , DNA Polymerase gamma , DNA-Directed DNA Polymerase/metabolism , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Exodeoxyribonucleases/metabolism , Humans , Protein Binding , Protein Stability , Protein Structure, Tertiary , Temperature , Tumor Suppressor Protein p53/chemistryABSTRACT
In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.
Subject(s)
DNA Damage/genetics , DNA Fingerprinting/methods , DNA-Directed DNA Polymerase/chemistry , DNA/genetics , Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods , Specimen Handling/methods , DNA-Directed DNA Polymerase/genetics , Forensic Genetics/methods , FossilsABSTRACT
Hydrophobic base analogues (HBAs) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. While extensive synthetic efforts have yielded examples of HBAs with favorable substrate properties, their discovery has remained challenging. Here we describe a complementary strategy for improving HBA substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (CSR) with the archetypal HBA 5-nitroindole (d5NI) and its derivative 5-nitroindole-3-carboxamide (d5NIC) as selection substrates. Starting from a repertoire of chimeric polymerases generated by molecular breeding of DNA polymerase genes from the genus Thermus, we isolated a polymerase (5D4) with a generically enhanced ability to utilize HBAs. The selected polymerase. 5D4 was able to form and extend d5NI and d5NIC (d5NI(C)) self-pairs as well as d5NI(C) heteropairs with all four bases with efficiencies approaching, or exceeding, those of the cognate Watson-Crick pairs, despite significant distortions caused by the intercalation of the d5NI(C) heterocycles into the opposing strand base stack, as shown by nuclear magnetic resonance spectroscopy (NMR). Unlike Taq polymerase, 5D4 was also able to extend HBA pairs such as Pyrene: varphi (abasic site), d5NI: varphi, and isocarbostyril (ICS): 7-azaindole (7AI), allowed bypass of a chemically diverse spectrum of HBAs, and enabled PCR amplification with primers comprising multiple d5NI(C)-substitutions, while maintaining high levels of catalytic activity and fidelity. The selected polymerase 5D4 promises to expand the range of nucleobase analogues amenable to replication and should find numerous applications, including the synthesis and replication of nucleic acid polymers with expanded chemical and functional diversity.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , DNA/chemistry , DNA/metabolism , Hydrophobic and Hydrophilic Interactions , Amides/chemistry , Bacteria/enzymology , Base Pairing , Base Sequence , DNA/genetics , DNA Primers/chemistry , DNA Primers/metabolism , DNA-Directed DNA Polymerase/chemistry , Indoles/chemistry , Kinetics , Models, Molecular , Protein Conformation , Substrate SpecificityABSTRACT
Universal bases hybridize with all other natural DNA or RNA bases, and have applications in PCR and sequencing. We have analysed by nuclear magnetic resonance spectroscopy the structure and dynamics of three DNA oligonucleotides containing the universal base analogues 5-nitroindole and 5-nitroindole-3-carboxamide. In all systems studied, both the 5-nitroindole nucleotide and the opposing nucleotide adopt a standard anti conformation and are fully stacked within the DNA duplex. The 5-nitroindole bases do not base pair with the nucleotide opposite them, but intercalate between this base and an adjacent Watson-Crick pair. In spite of their smooth accommodation within the DNA double-helix, the 5-nitroindole-containing duplexes exist as a dynamic mixture of two different stacking configurations exchanging fast on the chemical shift timescale. These configurations depend on the relative intercalating positions of the universal base and the opposing base, and their exchange implies nucleotide opening motions on the millisecond time range. The structure of these nitroindole-containing duplexes explains the mechanism by which these artificial moieties behave as universal bases.
Subject(s)
DNA/chemistry , Indoles/chemistry , Models, Molecular , Adenine/chemistry , Base Pairing , Nuclear Magnetic Resonance, Biomolecular , Oligodeoxyribonucleotides/chemistry , Solutions , Temperature , Thymine/chemistryABSTRACT
We have analysed the influence of neighbouring base sequences on the mutagenesis induced by 7,8-dihydro-8-oxoguanine (8-oxoG or G(o)), a typical oxidative lesion of DNA, using the yeast oligonucleotide transformation technique. Two oligonucleotides, oligo-CCG(o) and oligo-CGG(o), each possessing a single 8-oxoG residue and represented by the sequences 5'-CCG(o)-3' and 5'-CGG(o)-3', respectively, were introduced into a chromosome of Saccharomyces cerevisiae and their mutagenic potentials were compared. In a wild-type strain, 8-oxoG showed very weak mutagenic potential in both cases. However, the lesion in 5'-CCG(o)-3' can cause efficient G-to-T transversion in a strain lacking the rad30 gene which encodes yeast DNA polymerase eta (Ypoleta). To explore the properties associated with this translesion synthesis (TLS), the same two oligonucleotides possessing an 8-oxoG were used as templates for a standing-start primer extension assay, and the nucleotide incorporation opposite 8-oxoG was investigated. We found that dATP incorporation opposite 8-oxoG with Ypoleta was low for both sequences. In particular, very low dATP incorporation was observed for the 5'-CCG(o)-3' sequence. These results account for the efficient inhibition of mutagenesis by Ypoleta. TLS plays an important role in one DNA sequence in terms of avoiding mutagenesis induced by 8-oxoG in yeast. In contrast, human yeast DNA polymerase eta showed higher dATP incorporation rates even with the 5'-CCG(o)-3' sequence.
Subject(s)
Guanine/analogs & derivatives , Mutagenesis , Saccharomyces cerevisiae/drug effects , Base Sequence , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Guanine/toxicity , Molecular Sequence Data , Mutation , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolismABSTRACT
The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O(6)-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O(6)-MeG:T, O(6)-MeG:C, O(6)-EtG:T, O(6)-EtG:C, G:T and G:C. Dissociation constants (K(d)), which reflect the strength of binding, followed the order G:T->O(6)-MeG:T->O(6)-EtG:T-=O(6)-EtG:C-> or =O(6)-MeG:C->G:C. These results suggest that a thymine base paired with O(6)-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O(6)-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.
Subject(s)
Escherichia coli Proteins/metabolism , MutS DNA Mismatch-Binding Protein/metabolism , Mutation , Oligonucleotides/metabolism , Alkylating Agents/pharmacology , Alkylation , Base Sequence , Gene Frequency , Guanine/metabolism , Molecular Sequence DataABSTRACT
We have studied the mutagenic properties of ribonucleotide analogues by reverse transcription to understand their potential as antiretroviral agents by mutagenesis of the viral genome. The templating properties of nucleotide analogues including 6-(beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c](1,2)oxazin-7-one, N4-hydroxycytidine, N4-methoxycytidine, N4-methylcytidine and 4-semicarbazidocytidine, which have been reported to exhibit ambiguous base pairing properties, were examined. We have synthesized RNA templates using T3 RNA polymerase, and investigated the specificity of the incorporation of deoxyribonucleoside triphosphates opposite these cytidine analogues in RNA by HIV and AMV reverse transcriptases. Except for N4-methylcytidine, both enzymes incorporated both dAMP and dGMP opposite these analogues in RNA. This indicates that they would be highly mutagenic if present in viral RNA. To study the basis of the differences among the analogues in the incorporation ratios of dAMP to dGMP, we have carried out kinetic analysis of incorporation opposite the analogues at a defined position in RNA templates. In addition, we examined whether the triphosphates of these analogues were incorporated competitively into RNA by human RNA polymerase II. Our present data supports the view that these cytidine analogues are mutagenic when incorporated into RNA, and that they may therefore be considered as candidates for antiviral agents by causing mutations to the retroviral genome.
Subject(s)
Anti-Retroviral Agents/chemistry , Cytidine/analogs & derivatives , Mutagenesis , Reverse Transcription , Ribonucleotides/chemistry , Cytidine/chemistry , DNA-Directed RNA Polymerases/metabolism , Deoxyribonucleotides/metabolism , HIV Reverse Transcriptase/metabolism , HeLa Cells , Humans , Kinetics , RNA Polymerase II/metabolism , Retroviridae/genetics , Templates, GeneticABSTRACT
DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide- (NTP) and deoxyribonucleotide-triphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2'-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2'-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes.
Subject(s)
DNA Polymerase I/metabolism , DNA-Directed RNA Polymerases/metabolism , Directed Molecular Evolution , RNA-Directed DNA Polymerase/metabolism , Taq Polymerase/metabolism , Amino Acid Sequence , DNA/biosynthesis , DNA Polymerase I/genetics , DNA-Directed RNA Polymerases/genetics , Deoxyribonucleotides/metabolism , Molecular Sequence Data , Mutation , Polymerase Chain Reaction , RNA/biosynthesis , RNA-Directed DNA Polymerase/genetics , Ribonucleotides/metabolism , Substrate Specificity , Taq Polymerase/geneticsABSTRACT
DNA polymerases recognize their substrates with exceptionally high specificity, restricting the use of unnatural nucleotides and the applications they enable. We describe a strategy to expand the substrate range of polymerases. By selecting for the extension of distorting 3' mismatches, we obtained mutants of Taq DNA polymerase that not only promiscuously extended mismatches, but had acquired a generic ability to process a diverse range of noncanonical substrates while maintaining high catalytic turnover, processivity and fidelity. Unlike the wild-type enzyme, they bypassed blocking lesions such as an abasic site, a thymidine dimer or the base analog 5-nitroindol and performed PCR amplification with complete substitution of all four nucleotide triphosphates with phosphorothioates or the substitution of one with the equivalent fluorescent dye-labeled nucleotide triphosphate. Such 'unfussy' polymerases have immediate utility, as we demonstrate by the generation of microarray probes with up to 20-fold brighter fluorescence.
Subject(s)
Directed Molecular Evolution/methods , Taq Polymerase/genetics , Base Pair Mismatch/genetics , Biotin/chemistry , DNA/chemistry , DNA/genetics , DNA Mutational Analysis , DNA Probes/chemical synthesis , DNA Probes/chemistry , Deoxyadenine Nucleotides/chemistry , Deoxyadenine Nucleotides/metabolism , Deoxyguanine Nucleotides/chemistry , Deoxyguanine Nucleotides/metabolism , Deoxyribose/chemistry , Deoxyuracil Nucleotides/chemistry , Deoxyuracil Nucleotides/metabolism , Fluorescein-5-isothiocyanate/chemistry , Indoles/chemistry , Kinetics , Microarray Analysis , Models, Molecular , Mutation/genetics , Point Mutation/genetics , Polymerase Chain Reaction , Pyrimidine Dimers/chemistry , Rhodamines/chemistry , Sequence Analysis, DNA , Substrate Specificity , Taq Polymerase/chemistry , Taq Polymerase/metabolism , Thionucleotides/chemistry , Thionucleotides/metabolismABSTRACT
A library of N6-hydroxy-, methoxy-, or amino-adenosine analogs was prepared and screened for anti-malarial properties. We found three compounds that possess anti-plasmodial activity in the low micromolar range against the multi-drug resistant VS1 strain, namely N6-hydroxy-9H-purin-6-amine (IC50 5.57 micro M), 2-amino-N6-amino-adenosine (IC50 12.2 micro M), and 2-amino-N6-amino-N6-methyladenosine (IC50 0.29 micro M). More importantly, the compounds were non-toxic, with 2-amino-N6-amino-N6-methyladenosine showing a selectivity index of 5008.
Subject(s)
Antimalarials/pharmacology , Nitrogen/chemistry , Plasmodium falciparum/drug effects , Purines/pharmacology , Animals , Antimalarials/chemistry , Purines/chemistryABSTRACT
We have analyzed the mutagenic specificity of an abasic site in DNA using the yeast oligonucleotide transformation assay. Oligonucleotides containing an abasic site or its analog were introduced into B7528 or its derivatives, and nucleotide incorporation opposite abasic sites was analyzed. Cytosine was most frequently incorporated opposite a natural abasic site (O) ('C-rule'), followed by thymine. Deletion of REV1 decreased the transformation efficiency and the incorporation of cytosine nearly to a background level. In contrast, deletion of RAD30 did not affect them. We compared the mutagenic specificity with that of a tetrahydrofuran abasic site (F), an abasic analog used widely. Its mutation spectrum was clearly different from that of O. Adenine, not cytosine, was most favorably incorporated. However, deletion of REV1 decreased the transformation efficiency with F-containing oligonucleotide as in the case of O. These results suggest that the bypass mechanism of F is different from that of O, although the bypasses in both cases are dependent on REV1. We also found that the mutagenic specificity of F can be affected by not only the adjacent bases, but also a base located two positions away from F.
Subject(s)
DNA/chemistry , Furans/chemistry , Mutagenesis , Saccharomyces cerevisiae/genetics , Models, Genetic , Mutation , Oligonucleotides/chemistry , Oligonucleotides/genetics , Transformation, Genetic , Uracil/chemistryABSTRACT
Deoxyribosyl-dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is a potent mutagenic deoxycytidine-derived base analogue capable of pairing with both A and G, thereby causing G. C --> A. T and A. T --> G. C transition mutations. We have found that the Escherichia coli DNA mismatch-repair system can protect cells against this mutagenic action. At a low dose, dP is much more mutagenic in mismatch-repair-defective mutH, mutL, and mutS strains than in a wild-type strain. At higher doses, the difference between the wild-type and the mutator strains becomes small, indicative of saturation of mismatch repair. Introduction of a plasmid containing the E. coli mutL(+) gene significantly reduces dP-induced mutagenesis. Together, the results indicate that the mismatch-repair system can remove dP-induced replication errors, but that its capacity to remove dP-containing mismatches can readily be saturated. When cells are cultured at high dP concentration, mutant frequencies reach exceptionally high levels and viable cell counts are reduced. The observations are consistent with a hypothesis in which dP-induced cell killing and growth impairment result from excess mutations (error catastrophe), as previously observed spontaneously in proofreading-deficient mutD (dnaQ) strains.
Subject(s)
Base Pair Mismatch , DNA Repair/physiology , Escherichia coli/genetics , Deoxycytidine/genetics , Escherichia coli/physiology , MutagenesisABSTRACT
Rev1p in yeast is essential for the translesion of abasic sites and 6-4 photoproducts. It plays a role as a translesion polymerase, but also supports translesion catalyzed by other polymerases. The protein has two domains, BRCT and Y-family polymerase. A point mutation in the BRCT domain is known to abolish the second function. In the present research, we have studied the effects of deletion of the BRCT domain and a point mutation at the two amino acids in the putative polymerase active center. We have introduced an abasic site, its tetrahydrofuran analog, and a 6-4 thymine-thymine photoproduct using the oligonucleotide transformation assay. Translesion efficiencies were estimated from the transforming activities of the oligonucleotides with a lesion, and the mutation spectra were analyzed by DNA sequencing of the transformants. Results showed that the lack of the BRCT domain reduced translesion efficiencies, but that substantial translesion synthesis took place. The mutation spectra of the lesions were not greatly affected. Therefore, the BRCT domain may be important, but dispensable for translesion synthesis. In contrast, the polymerase mutation, rev1AA, has only small effects on the translesion efficiencies, but the mutation spectra were greatly affected; the incorporation of dCMP opposite the lesions was specifically lost. This clearly shows that the polymerase domain is responsible for the dCMP incorporation. The effect of Poleta was also analyzed. From all the results DNA polymerases other than these two translesion polymerases, too, seem to initiate the translesion synthesis.
Subject(s)
DNA Damage , DNA-Directed DNA Polymerase/metabolism , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , DNA/biosynthesis , DNA Mutational Analysis , DNA, Fungal , Models, Biological , Nucleotidyltransferases/genetics , Point Mutation , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence DeletionABSTRACT
We have studied mutagenic specificities of DNA lesions in vivo in yeast CYC1 oligonucleotide transformation assay. We introduced two lesions into oligonucleotides. One was a nucleoside analog, 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one 2'-deoxyriboside (dP), which is highly mutagenic to bacteria. It is supposed to be a miscoding, but otherwise good template for DNA polymerases. The other lesion was the TT pyrimidine(6-4)pyrimidone photoproduct, one of the typical UV lesions, which blocks DNA replication. These oligonucleotides were used to transform yeast cyc1 mutants with ochre nonsense mutation to Cyc1+. As expected from its templating properties in vitro, the transforming activity of dP-containing oligonucleotides was similar to those of unmodified oligonucleotides. Results indicated that dP may direct incorporation of guanine and adenine at a ratio of 1:20 or more in vivo. An oligonucleotide containing the photoproduct showed the transforming activity of as low as 3-5% of that of the corresponding unmodified oligonucleotide. This bypass absolutely required REV1 gene. The sequence analysis of the transformants has shown that the lesion was read as TT and TC at a ratio of 3:7, indicating its high mutagenic potential.
Subject(s)
DNA Damage , DNA, Fungal/genetics , Mutagens/pharmacology , Oligonucleotides/pharmacology , Saccharomyces cerevisiae/genetics , Base Sequence , DNA PrimersABSTRACT
Sequence-specific oligonucleotide hybridization (SSOH, 'dot-blotting') is a widely employed method of typing single nucleotide polymorphisms (SNPs), but it is often compromised by lack of allelic differentiation. We describe a novel improvement to SSOH that incorporates an additional mismatch into the oligonucleotide probe using the universal base analogue 3-nitropyrrole. This method greatly increases allelic differentiation compared to standard SSOH where oligonucleotides contain only SNP-defining base changes. Moreover, stringency of the hybridisation is predictably maintained over a wide range of temperatures, which can be calculated empirically, thus facilitating the genotyping of multiple SNPs using similar conditions. This improved method increases the usefulness of hybridisation-based methods of rapid genotyping of SNPs and may have implications for array methodologies.
Subject(s)
Nucleic Acid Hybridization/methods , Oligonucleotide Probes/chemistry , Polymorphism, Single Nucleotide , Pyrroles/chemistry , Alleles , Base Pair Mismatch , Oligonucleotide Probes/chemical synthesis , Transition TemperatureABSTRACT
Addition of poly(ADP-ribose) (PAR) is an important post-translational modification in higher eukaryotes. Several DNA repair and checkpoint proteins possess specific PAR-binding zinc-finger (PBZ) modules critical for function. Here, we present solution structures of the two PBZ modules of aprataxin and PNK-like factor (APLF), revealing a novel type of zinc finger. By combining in vivo PAR-binding data with NMR interaction data using PAR fragments, we propose a structural basis for PBZ-PAR recognition.
Subject(s)
Phosphoproteins/chemistry , Phosphoproteins/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Amino Acid Sequence , DNA-(Apurinic or Apyrimidinic Site) Lyase , Humans , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Poly-ADP-Ribose Binding Proteins , Protein Binding , Protein Structure, Tertiary , Zinc FingersABSTRACT
DNA is not only a repository of genetic information for life, it is also a unique polymer with remarkable properties: it associates according to well-defined rules, it can be assembled into diverse nanostructures of defined geometry, it can be evolved to bind ligands and catalyse chemical reactions and it can serve as a supramolecular scaffold to arrange chemical groups in space. However, its chemical makeup is rather uniform and the physicochemical properties of the four canonical bases only span a narrow range. Much wider chemical diversity is accessible through solid-phase synthesis but oligomers are limited to <100 nucleotides and variations in chemistry can usually not be replicated and thus are not amenable to evolution. Recent advances in nucleic acid chemistry and polymerase engineering promise to bring the synthesis, replication and ultimately evolution of nucleic acid polymers with greatly expanded chemical diversity within our reach.
Subject(s)
Biopolymers/chemistry , DNA-Directed DNA Polymerase/chemistry , DNA/chemistry , DNA/physiology , Nanostructures/chemistry , Protein Engineering , DNA/genetics , Protein BindingABSTRACT
The total synthesis of a simple cell is in many ways the ultimate challenge in synthetic biology. Outlined eight years ago in a visionary article by Szostak et al. (J. W. Szostak, D. P. Bartel and P. L. Luisi, Nature, 2001, 409, 387), the chances of success seemed remote. However, recent progress in nucleic acid chemistry, directed evolution and membrane biophysics have brought the prospect of a simple synthetic cell with life-like properties such as growth, division, heredity and evolution within reach. Success in this area will not only revolutionize our understanding of abiogenesis but provide a fertile test-bed for models of prebiotic chemistry and early evolution. Last but not least, a robust "living" protocell may provide a versatile and safe chassis for embedding synthetic devices and systems.