Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates

Recent development of the long PCR technology has provided an invaluable tool in many areas of molecular biology. However, long PCR amplification fails whenever the DNA template is imperfectly preserved. We report that Escherichia coli exonuclease III, a major repair enzyme in bacteria, strikingly improves the long PCR amplification of damaged DNA templates. Escherichia coli exonuclease III permitted or improved long PCR amplification with DNA samples submitted to different in vitro treatments known to induce DNA strand breaks and/or apurinic/apyrimidinic (AP) sites, including high temperature (99°C), depurination at low pH and near-UV radiation. Exonuclease III also permitted or improved amplification with DNA samples that had been isolated several years ago by the phenol/chloroform method. Amelioration of long PCR amplification was achieved for PCR products ranging in size from 5 to 15.4 kb and with DNA target sequences located either within mitochondrial DNA or the nuclear genome. Exonuclease III increased the amplification of damaged templates using either rTth DNA polymerase alone or rTth plus Vent DNA polymerases or Taq plus Pwo DNA polymerases. However, exonuclease III could not improve PCR amplification from extensively damaged DNA samples. In conclusion, supplementation of long PCR mixes with E.coli exonuclease III may represent a major technical advance whenever DNA samples have been partly damaged during isolation or subsequent storage.     

Binding of Escherichia coli DNA photolyase to UV-irradiated DNA

Escherichia coli DNA photolyase is a flavoprotein which catalyzes the photomonomerization of pyrimidine dimers produced in DNA by UV irradiation. In vivo, the enzyme acts by a two-step mechanism: it binds to dimer-containing DNA in a light-independent reaction and upon exposure to 300-500-nm light breaks the cyclobutane ring and dissociates from the substrate. Using photolyase purified to homogeneity, we have investigated in vitro the first step of the reaction, DNA binding; enzyme-DNA complex formation was quantitated by the nitrocellulose filter binding assay. We find that the enzyme binds specifically to UV-irradiated DNA regardless of whether the DNA is in the superhelical, open circular, or linear form or whether the DNA is single or double stranded. The binding reaction is optimum at a NaCl concentration of 125 mM and at pH 7.5. Although photolyase is retained by the nitrocellulose filters with near 100% efficiency, the binding efficiency of a single enzyme-substrate complex is about 0.34. The complexes can be dissociated by exposing them to photoreactivating light either in solution or on the filter.     

Melanin-based high-throughput screen for L-tyrosine production in Escherichia coli

We present the development of a simple, high-throughput screen for identifying bacterial strains capable of L-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link L-tyrosine production in Escherichia coli with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient for increasing both the rates of synthesis and the final titers of melanin. Furthermore, a strong linear correlation between extracellular L-tyrosine content and melanin formation was observed by use of this new medium formulation. A selection strategy that utilizes these findings has been developed and has been shown to be effective in screening large combinatorial libraries in the search for L-tyrosine-overproducing strains.     

A genetic screen to identify sequences that mediate protein oligomerization in Escherichia coli

Many proteins assemble as oligomeric complexes and in several cases a distinct domain mediates the interaction between the subunits. The identification of new oligomerization modules is relevant to comprehend both the architecture and the evolution of protein sequences and also for protein engineering applications. Using the bacteriophage lambda repressor dimerization assay, we searched Escherichia coli genomic libraries for sequences able to mediate protein oligomerization in vivo. We identified short peptides that can substitute very effectively the dimerizing domain of the repressor. Most of these peptides belong to open reading frames that are normally not expressed in the bacterial cell.     

Escherichia coli responses to a single DNA adduct

To study the mechanisms by which Escherichia coli modulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various E. coli pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1, N(6)-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional recA, recF, recO, and recR genes, suggesting the mechanism to be daughter strand gap repair; (iii) the ruvABC genes or the recG gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed.     

Y-family DNA polymerases in Escherichia coli

The observation that mutations in the Escherichia coli genes umuC+ and umuD+ abolish mutagenesis induced by UV light strongly supported the counterintuitive notion that such mutagenesis is an active rather than passive process. Genetic and biochemical studies have revealed that umuC+ and its homolog dinB+ encode novel DNA polymerases with the ability to catalyze synthesis past DNA lesions that otherwise stall replication--a process termed translesion synthesis (TLS). Similar polymerases have been identified in nearly all organisms, constituting a new enzyme superfamily. Although typically viewed as unfaithful copiers of DNA, recent studies suggest that certain TLS polymerases can perform proficient and moderately accurate bypass of particular types of DNA damage. Moreover, various cellular factors can modulate their activity and mutagenic potential.     

多基因在大肠杆菌中的共表达策略

多基因共表达在多领域有重要的应用价值,大肠杆菌共表达系统包括多顺反子系统和双质粒系统,两者各有优缺点。多顺反子系统不需外界2种抗生素的同时存在,但操作较为复杂。通常认为,具有相同复制子的质粒是不相容的,但近来的实验表明,在双抗生素的选择压力下,不相容的双质粒系统也能稳定传代,且操作简单,周期较短。双质粒系统已经应用于生产、医学等各个领域。     

Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome

Recombinant DNA libraries generated in vitro should in theory contain all of the sequences of the genomes from which they are derived. However, the literature is dotted with reports of sequences that cannot be recovered, are under-represented, or are highly unstable. In particular, long palindromic nucleotide sequences of perfect or near-perfect symmetry are either lethal to the vector or suffer deletions or other rearrangements that remove symmetry [Collins, Cold Spring Harbor Symp. Quant. Biol. 45 (1981) 409-416; Collins et al., Gene 19 (1982) 139-146; Hagan and Warren, Gene 24 (1983) 317-326]. We report here that mutation of a single gene, namely sbcC, can overcome this inviability and allow for the stable propagation of a 571-bp nearly perfect palindrome in Escherichia coli. This has implications for the choice of strains used for the recovery and analysis of cloned nucleotide sequences.     

Corynebacterium glutamicum DNA is subjected to methylation-restriction in Escherichia coli

Abstract Efficient electroporation of Escherichia coli with plasmid DNA isolated from Corynebacterium glutamicum depends on the use of Mcr-deficient E. coli strains. The transformation frequency increased nearly 800-fold when the Mcr-deficient E. coli DH5αMCR was used instead of E. coli DH5α. We used E. coli strains with different mutations in the methyl-specific restriction systems to show that McrBC-deficiency is sufficient to generate this effect. The results imply that C. glutamicum DNA contains methylcytosine in specific sequences recognized by the E. coli McrBC system.     

Characterization of unintegrated retroviral DNA with long terminal repeat-associated cell-derived inserts.

We have used a replication-competent shuttle vector based on the genome of Rous sarcoma virus to characterize genomic rearrangements that occur during retrovirus replication. The strategy involved cloning circular DNA that was generated during an acute infection. While analyzing a class of retroviral DNA clones that are greater than full length, we found several clones which had acquired nonviral inserts in positions adjacent to the long terminal repeats (LTRs). There appear to be two distinct mechanisms leading to the incorporation of cellular sequences into these clones. Three of the molecules contain a cell-derived insert at the circle junction site between two LTR units. Two of these molecules appear to be the results of abortive integration attempts, because of which, in each case, one of the LTRs is missing 2 bases at its junction with the cell-derived insert. In the third clone, pNO220, the cellular sequences are flanked by an inappropriately placed copy of the tRNA primer-binding site on one side and a partial copy of the U3 sequence as part of the LTR on the other side. A fourth molecule we characterized, pMD96, has a single LTR with a U5-bounded deletion of viral sequences spanning gag and pol, with cell-derived sequences inserted at the site of the deletion; its origin may be related mechanistically to pNO220. Sequence analysis indicates that all of the cellular inserts were derived from the cell line used for the acute infection rather than from sequences carried into the cell as part of the virus particle. Northern (RNA) analysis of cellular RNA demonstrated that the cell-derived sequences of two clones, pNO220 and pMD96, were expressed as polyadenylated RNA in uninfected cells. One mechanism for the joining of viral and cellular sequences suggested by the structures of pNO220 and pMD96 is recombination occurring during viral DNA synthesis, with cellular RNA serving as the template for the acquisition of cellular sequences.