The XPF-ERCC1 endonuclease and homologous recombination contribute to the repair of minor groove DNA interstrand crosslinks inmammalian cells produced by the pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136

SJG-136, a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, is a highly efficient interstrand crosslinking agent that reacts with guanine bases in a 5'-GATC-3' sequence in the DNA minor groove. SJG-136 crosslinks form rapidly and persist compared to those produced by conventional crosslinking agents such as nitrogen mustard, melphalan or cisplatin which bind in the DNA major groove. A panel of Chinese hamster ovary (CHO) cells with defined defects in specific DNA repair pathways were exposed to the bi-functional agents SJG-136 and melphalan, and to their mono-functional analogues mmy-SJG and mono-functional melphalan. SJG-136 was >100 times more cytotoxic than melphalan, and the bi-functional agents were much more cytotoxic than their respective mono-functional analogues. Cellular sensitivity of both SJG-136 and melphalan was dependent on the XPF-ERCC1 heterodimer, and homologous recombination repair factors XRCC2 and XRCC3. The relative level of sensitivity of these repair mutant cell lines to SJG-136 was, however, significantly less than with major groove crosslinking agents. In contrast to melphalan, there was no clear correlation between sensitivity to SJG-136 and crosslink unhooking capacity measured using a modified comet assay. Furthermore, repair of SJG-136 crosslinks did not involve the formation of DNA double-strand breaks. SJG-136 cytotoxicity is likely to result from the poor recognition of DNA damage by repair proteins resulting in the slow repair of both mono-adducts and more importantly crosslinks in the minor groove

Next-generation plastic degrading enzymes

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Next-generation plastic degrading enzymes

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Selection of bacteriophage λ integrases with altered recombination specificity by in vitro compartmentalization

In vitro compartmentalization (IVC) was employed for the first time to select for novel bacteriophage λ integrase variants displaying significantly enhanced recombination activity on a non-cognate target DNA sequence. These variants displayed up to 9-fold increased recombination activity over the parental enzyme, and one mutant recombined the chosen non-cognate substrate more efficiently than the parental enzyme recombined the wild-type DNA substrate. The in vitro specificity phenotype extended to the intracellular recombination of episomal vectors in HEK293 cells. Surprisingly, mutations conferring the strongest phenotype do not occur in the λ integrase core-binding domain, which is known to interact directly with cognate target sequences. Instead, they locate to the N-terminal domain which allosterically modulates integrase activity, highlighting a previously unknown role for this domain in directing integrase specificity. The method we describe provides a robust, completely in vitro platform for the development of novel integrase reagent tools for in vitro DNA manipulation and other biotechnological applications

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. 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

Indole and azaindole halogenation catalyzed by the RebH enzyme variant 3-LSR utilizing co-purified E. coli reductase

Biocatalytic C-H halogenation is becoming increasingly attractive due to excellent catalyst-controlled selectivity and environmentally benign reaction conditions. Significant efforts have been made on enzymatic halogenation of industrial arenes in a cost-effective manner. Here we report an unprecedented enzymatic halogenation of a panel of industrially important indole, azaindole and anthranilamide derivatives using a thermostable RebH variant without addition of any external flavin reductase enzyme. The reactions were catalyzed by the RebH variant 3-LSR enzyme with the help of a co-purified E. coli reductase identified as alkyl hydroperoxide reductase F (AhpF)

Cell-free selection of RNA-binding proteins using in vitro compartmentalization

RNA-binding proteins (RBPs) perform many essential functions in the post-transcriptional control of gene expression. If we were able to engineer RBPs with new specificity, it would also become possible to develop new tools to control and investigate gene expression pathways. Molecular evolution methods such as phage display have been introduced to achieve this goal, but the large interface between these proteins and RNA relative to the size of library that can be constructed limits the efficacy of this method. In order to increase the diversity of libraries used for selection of RBPs, we applied the emulsion-based in vitro compartmentalization (IVC) method to select RBPs with defined specificity. A new approach was developed to link genotype and phenotype by fusing the target RBP to zinc finger proteins (ZFPs) that bind to a cognate DNA sequence inserted upstream of the promoter. The expressed fusion protein (ZFP–RBP) binds to its encoding DNA with high affinity via the ZFP target-binding site. After breaking the emulsion, the RBP can be selected based on its affinity for a biotinylated RNA bait. We demonstrate the effectiveness of this method that should enable the selection of RBPs with new specificity or improved affinity

Long-range, high-throughput haplotype determination via haplotype-fusion PCR and ligation haplotyping

Ligation Haplotyping is a robust, novel method for experimental determination of haplotypes over long distances, which can be applied to assaying both sequence and structural variation. The simplicity and efficacy of the method for genotyping large chromosomal rearrangements and haplotyping SNPs over long distances make it a valuable and powerful addition to the methodological repertoire, which will be beneficial to studies of population genetics and evolution, disease association and inheritance, and genomic variation. We illustrate the versatility of the method both by genotyping a Yp paracentric inversion, found in ∼60% of Northwest European males, that strongly influences the germline rate of infertility-causing XY translocations and by haplotyping two autosomal SNPs that lie 16.4 kb apart on chromosome 7, and which influence an individual's susceptibility to systemic lupus erythematosus

Iminodiacetic-phosphoramidates as metabolic prototypes for diversifying nucleic acid polymerization in vivo

Previous studies in our laboratory proved that certain functional groups are able to mimic the pyrophosphate moiety and act as leaving groups in the enzymatic polymerization of deoxyribonucleic acids by HIV-1 reverse transcriptase. When the potential leaving group possesses two carboxylic acid moieties linked to the nucleoside via a phosphoramidate bond, it is efficiently recognized by this error-prone enzyme, resulting in nucleotide incorporation into DNA. Here, we present a new efficient alternative leaving group, iminodiacetic acid, which displays enhanced kinetics and an enhanced elongation capacity compared to previous results obtained with amino acid deoxyadenosine phosphoramidates. Iminodiacetic acid phosphoramidate of deoxyadenosine monophosphate (IDA-dAMP) is processed by HIV-1 RT as a substrate for single nucleotide incorporation and displays a typical Michaelis–Menten kinetic profile. This novel substrate also proved to be successful in primer strand elongation of a seven-base template overhang. Modelling of this new substrate in the active site of the enzyme revealed that the interactions formed between the triphosphate moiety, magnesium ions and enzyme's residues could be different from those of the natural triphosphate substrate and is likely to involve additional amino acid residues. Preliminary testing for a potential metabolic accessibility lets us to envision its possible use in an orthogonal system for nucleic acid synthesis that would not influence or be influenced by genetic information from the outside