Synthetic polymers and their potential as genetic materials.

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.

Structure of the 70S ribosome bound to release factor 2 and a substrate analog provides insights into catalysis of peptide release.

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.

Solution structures of the two PBZ domains from human APLF and their interaction with poly(ADP-ribose).

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.

Evolving a polymerase for hydrophobic base analogues.

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.

Polymerase engineering: towards the encoded synthesis of unnatural biopolymers.

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.

Darwinian chemistry: towards the synthesis of a simple cell.

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.

Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome.

Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. In addition, although past structures of the 50S subunit have found no ordered proteins at the PTC, biochemical evidence suggests that specific proteins are capable of interacting with the 3' ends of tRNA ligands.

Physical and functional interactions between human mitochondrial single-stranded DNA-binding protein and tumour suppressor p53.

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.

Translesion synthesis across the (6-4) photoproduct and its Dewar valence isomer by the Y-family and engineered DNA polymerases.

We analyzed the translesion synthesis across the UV-induced lesions, the (6-4) photoproduct and its Dewar valence isomer, by using human DNA polymerases eta and iota in vitro. The primer extension experiments revealed that pol eta tended to incorporate dG opposite the 3' component of both lesions, but the incorporation efficiency for the Dewar isomer was higher than that for the (6-4) photoproduct. On the other hand, pol iota was likely to incorporate dA opposite the 3' components of the (6-4) photoproduct and its Dewar isomer with a similar efficiency.

Influence of neighbouring base sequences on the mutagenesis induced by 7,8-dihydro-8-oxoguanine in yeast.

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.

Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency.

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.

Lethal mutagenesis of picornaviruses with N-6-modified purine nucleoside analogues.

RNA viruses exhibit extraordinarily high mutation rates during genome replication. Nonnatural ribonucleosides that can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication have been explored as antiviral agents acting through lethal mutagenesis. We have synthesized novel N-6-substituted purine analogues with ambiguous incorporation characteristics due to tautomerization of the nucleobase.

In vitro anti-malarial activity of N6-modified purine analogs.

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.

Oligonucleotide transformation for the study of mutagenic specificities of DNA lesions in yeast.

We developed a method for the analyzing mutagenic potential of DNA damage based on the oligonucleotide transformation technique in yeast. Using this assay we have analyzed mutagenic specificities of various DNA lesions. In the present study, we analyzed the mutagenic properties of 2-hydroxyadenine and 5-hydroxycytosine in yeast.

Nucleotide incorporation against 7,8-dihydro-8-oxoguanine is influenced by neighboring base sequences in TLS DNA polymerase reaction.

7,8-Dihydro-8-oxoguanine (8-oxoG) is a well-known oxidative lesion in DNA and is related to carcinogenesis and ageing processes. Misincorporation of dATP opposite to 8-oxoG leads to G --> T transversion mutations. DNA sequence has been proved as an important factor influencing the replication and enzymatic repair of various types of damages.

Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR.

The sequence saturation mutagenesis (SeSaM) method has been advanced to a random mutagenesis method with adjustable mutational biases. SeSaM offers, for example, a bias that is complementary to error-prone (ep) PCR and is enriched in transversions (SeSaM-Tv(+)). dNTP alpha S and three degenerate bases (P, K and I) are used to control mutational bias flexibly.

Lethal mutagenesis of poliovirus mediated by a mutagenic pyrimidine analogue.

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome.

Molecular breeding of polymerases for amplification of ancient DNA.

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.

Anti-malarial activity of N6-modified purine analogues.

Plasmodium falciparum causes one of the deadliest forms of malaria and resistance to the currently available drugs makes it imperative to develop new, safe and potent drugs. Parasites such as P. falciparum are unable to synthesise purines de novo and to this end often have multiple purine uptake and salvage systems.

Solution structure and dynamics of DNA duplexes containing the universal base analogues 5-nitroindole and 5-nitroindole 3-carboxamide.

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.

Mutagenic properties of ribonucleotide analogues in reverse transcription with HIV and AMV reverse transcriptases.

Pyrimidine analogues, N4-hydroxycytosine (C(oh)), N4-methoxycytosine (C(mo)) and 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P) can form base pairs with both adenine and guanine. We examined the mutagenic properties of these ribonucleotide analogues in RNA in reverse transcription with HIV and AMV reverse transcriptases. Both reverse transcriptases incorporated dATP and dGTP opposite these analogues in RNA.

UVA-induced degradation of 8-hydroxyguanine in oligonucleotide and the effect on its activities in yeast oligonucleotide transformation assay.

UVA-induced conversion of 8-hydroxyguanine in oligonucleotides was studied. By irradiation with 334 nm UVA light, 8-hydroxyguanine was completely changed to unknown compounds. Monomeric nucleoside may be much less labile to UVA.

Template properties of mutagenic cytosine analogues in reverse transcription.

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.

Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.

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.