Epitopes and active sites of the RecA protein.

The RecA protein is indispensable for homologous genetic recombination in Escherichia coli. This protein alone promotes the ATP-dependent formation of homologous joint molecules and their processing in vitro. Through the use of a set of anti-RecA protein mouse monoclonal IgGs, we have been attempting to divide the whole process into elementary steps to determine the basic functions of the protein. In order to correlate the basic functions with the active sites on the recA polypeptide, we located the epitopes for the anti-RecA protein-IgGs on the recA polypeptide by means of immunoblotting experiments and an enzyme-linked immunosorbent assay involving isolated proteolytic polypeptides or synthetic ones derived from various regions of the recA polypeptide. The epitopes for anti-RecA protein-IgGs ARM321 and ARM414, both of which are shown to inhibit the DNA-dependent ATP hydrolysis and the formation of homologous joints by the RecA protein, were found to be located between Thr89 and Glu127 and between Glu233 and Lys256, respectively, on the RecA polypeptide. IgG ARM193 had been shown to interfere with the protein-protein interaction between two RecA protein molecules, and ARM191 had been suggested to inhibit the binding of double-stranded DNA to the RecA protein. The epitopes for ARM193 and ARM191 were found to be located in a approximately 90-amino acid region at the C terminus. These results suggest the locations of the active sites and a functional core on the RecA polypeptide.     

An aerobic recA-, umuC-dependent pathway of spontaneous base-pair substitution mutagenesis in Escherichia coli

Antimutator alleles indentify genes whose normal products are involved in spontaneous mutagenesis pathways. Mutant alleles of the recA and umuC genes of Escherichia coli, whose wild-type alleles are components of the inducible SOS response, were shown to cause a decrease in the level of spontaneous mutagenesis. Using a series of chromosomal mutant trp alleles, which detect point mutations, as a reversion assay, it was shown that the reduction in mutagenesis is limited to base-pair substitutions. Within the limited number of sites than could be examined, transversions at AT sites were the favored substitutions. Frameshift mutagenesis was slightly enhanced by a mutant recA allele and unchanged by a mutant umuC allele. The wild-type recA and umuC genes are involved in the same mutagenic base-pair substitution pathway, designated "SOS-dependent spontaneous mutagenesis" (SDSM), since a recAumuC strain showed the same degree and specificity of antimutator activity as either single mutant strain. The SDSM pathway is active only in the presence of oxygen, since wild-type, recA, and umuC strains all show the same levels of reduced spontaneous mutagenesis anaerobically. The SDSM pathway can function in starving/stationary cells and may, or may not, be operative in actively dividing cultures. We suggest that, in wild-type cells, SDSM results from basal levels of SOS activity during DNA synthesis. Mutations may result from synthesis past cryptic DNA lesions (targeted mutagenesis) and/or from mispairings during synthesis with a normal DNA template (untargeted mutagenesis). Since it occurs in chromosomal genes of wild-type cells, SDSM may be biologically significant for isolates of natural enteric bacterial populations where extended starvation is often a common mode of existence.     

Method for gene replacement in Pseudomonas aeruginosa used in construction of recA mutants: recA-independent instability of alginate production.

The availability of a technique for site-directed mutagenesis by gene replacement provides a powerful tool for genetic analysis in any bacterial species. We report here a general technique for gene replacement in Pseudomonas aeruginosa. Genes on fragments of cloned P. aeruginosa DNA, altered by transposon mutagenesis, can be transduced into a recipient strain and can replace homologous genes in the P. aeruginosa genome. In this study we applied this technique to the construction of recA mutants of P. aeruginosa. A cloned segment of P. aeruginosa FRD1 DNA was isolated which encoded a protein analogous to the recA gene product of Escherichia coli. The P. aeruginosa recA gene was able to complement several defects associated with recA mutation in E. coli. Transposon Tn1 and Tn501 insertions in the cloned recA gene of P. aeruginosa were used to generate chromosomal recA mutants by gene replacement. These recA strains of P. aeruginosa were more sensitive to UV irradiation and methyl methane sulfonate and showed reduced recombination proficiency compared with the wild type. Also examined was the effect of recA mutations on the expression of alginate, a virulence trait. Alginate is a capsulelike polysaccharide associated with certain pulmonary infections, and its expression is typically unstable. The genetic mechanism responsible for the instability of alginate biosynthesis was shown to be recA independent.     

Monoclonal antibodies and mechanistic studies on recA protein.

A protein has various epitopes, and a monoclonal antibody specifically binds to the protein by recognizing 1 of the epitopes. This characteristic of the monoclonal antibody has opened various new approaches in a wide variety of research works. In studies about recA protein and its promoted various reactions relating to genetic recombination, anti-recA protein-monoclonal antibodies are very useful to analyse reaction mechanisms and to detect transition in the higher order-structure of the protein, as well as to measure the amounts of recA protein in vitro or in vivo and to identify the related proteins. In this article, we will review studies on recA protein in which monoclonal antibodies were used as major tools. By using anti-recA protein-monoclonal IgGs as specific inhibitors, the partial reactions of the homologous pairing and strand exchange promoted by recA protein were separated, and by use of a set of anti-recA protein IgGs the stages of activation of recA protein in the above reactions were discriminated.     

RecA protein of Escherichia coli has a third essential role in SOS mutator activity.

The DNA damage-inducible SOS response of Escherichia coli includes an error-prone translesion DNA replication activity responsible for SOS mutagenesis. In certain recA mutant strains, in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity. Like UV mutagenesis, SOS mutator activity requires the products of the umuDC operon and depends on RecA protein for at least two essential activities: facilitating cleavage of LexA repressor to derepress SOS genes and processing UmuD protein to produce a fragment (UmuD') that is active in mutagenesis. To determine whether RecA has an additional role in SOS mutator activity, spontaneous mutability (tryptophan dependence to independence) was measured in a family of nine lexA-defective strains, each having a different recA allele, transformed or not with a plasmid that overproduces either UmuD' alone or both UmuD' and UmuC. The magnitude of SOS mutator activity in these strains, which require neither of the two known roles of RecA protein, was strongly dependent on the particular recA allele that was present. We conclude that UmuD'C does not determine the mutation rate independently of RecA and that RecA has a third essential role in SOS mutator activity.     

Monoclonal antibodies with specific effects on partial activities of recA protein of Escherichia coli

recA protein of Escherichia coli promotes a wide variety of DNA reactions in vitro. Specific effectors of recA protein should be very useful in elucidating the mechanisms of these complex reactions. Six mouse hybridoma clones that secreted class G immunoglobulins specific to recA protein were obtained in three cell-fusion experiments. Five IgGs were purified by affinity chromatography. These monoclonal antibodies were characterized by examining their effects on the single-stranded DNA-dependent ATPase activity, negatively superhelical double-stranded DNA-dependent ATPase activity, and an activity in pairing negatively superhelical closed circular double-stranded DNA and homologous single-stranded DNA-fragments to form D-loops. These IgGs inhibited all, some, or one of these three activities, and from the spectra of their inhibitory effects they were classified into four groups. This classification suggests that each of the monoclonal antibodies binds to one of at least four antigenic determinants on recA protein and specifically inactivates one or more of the active centers on the protein. These monoclonal antibodies will be useful in analyzing the complex reactions promoted by recA protein.     

The Mycobacterium tuberculosis recA intein can be used in an ORFTRAP to select for open reading frames

The DNA repair protein RecA of Mycobacterium tuberculosis contains an intein, a self-splicing protein element. We have employed this Mtu recA intein to create a selection system for successful intein splicing by inserting it into a kanamycin-resistance gene so that functional antibiotic resistance can only be restored upon protein splicing. We then proceeded to develop an ORFTRAP, i.e., a selection system for the cloning of open reading frames (ORFs). The ORFTRAP exploits the self-splicing properties of inteins (which depend on full-length in-frame translation of a precursor protein) by allowing protein splicing to occur when DNA fragments encoding ORFs are inserted into the Mtu recA intein, whereas DNA fragments containing non-ORFs are selected against. Regions of the Mtu recA intein that tolerate the insertion of additional amino acids were identified by Bgl II linker scanning mutagenesis, and a respective construct was chosen as the ORFTRAP. To test the maximum insert size that could be cloned into ORFTRAP, DNA fragments of increasing length from the Listeria monocytogenes hly gene as well as a genomic library of Haemophilus influenzae were inserted and it was found that the longest permissive inserts were 425 bp and 251 bp, respectively. The H. influenzae ORFTRAP library also demonstrated the strength (strong selection power) and weakness (insertion of very small fragments) of the system. Further modifications should make the ORFTRAP useful for protein expression, epitope mapping, and antigen screening.     

Two-dimensional structure of the Opc invasin from Neisseria meningitidis

A two-dimensional structural model was devised for the Opc outer membrane protein invasin which contains 10 transmembrane strands and five surface-exposed loops. One continuous epitope recognized by three monoclonal antibodies was localized to the tip of loop 2 by synthetic peptides and site-directed mutagenesis while a second, discontinuous epitope recognized by a fourth antibody was localized to loops 4 and 5 by insertion mutagenesis. These monoclonal antibodies are bactericidal and inhibit adhesion and invasion. Most of the T-cell epitopes defined by Wiertz et al. (1996) were localized to the transmembrane strands. Oligonucleotides encoding a foreign epitope (∇) from Semliki Forest virus were inserted into Bgl II restriction sites created by site-directed mutagenesis. The ∇ epitopes inserted in all five predicted loops were recognized on the cell surface of live Escherichia coli bacteria by a monoclonal antibody and are exposed while ∇ epitopes in the N-terminus or three predicted turns were not. The results thus confirm important predictions of the model and define five permissive sites within surface-exposed loops which can be used to insert foreign epitopes.     

Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage.

Evidence is presented that recA functions which promote the SOS functions of mutagenesis, LexA protein proteolysis, and lambda cI repressor proteolysis are each genetically separable from the others. This separation was observed in recombination-proficient recA mutants and rec+ (F' recA56) heterodiploids. recA430, recA433, and recA435 mutants and recA+ (F' recA56) heterodiploids were inducible for only one or two of the three functions and defective for mutagenesis. recA80 and recA432 mutants were constitutively activated for two of the three functions in that these mutants did not have to be induced to express the functions. We propose that binding of RecA protein to damaged DNA and subsequent interaction with small inducer molecules gives rise to conformational changes in RecA protein. These changes promote surface-surface interactions with other target proteins, such as cI and LexA proteins. By this model, the recA mutants are likely to have incorrect amino acids substituted as sites in the RecA protein structure which affect surface regions required for protein-protein interactions. The constitutively activated mutants could likewise insert altered amino acids at sites in RecA which are involved in the activation of RecA protein by binding small molecules or polynucleotides which metabolically regulate RecA protein.     

Epitope mapping by solution NMR spectroscopy

Antibodies play an ever more prominent role in basic research as well as in the biotechnology and pharmaceutical sectors. Characterizing their epitopes, that is, the region that they recognize on their target molecule, is useful for purposes ranging from molecular biology research to vaccine design and intellectual property protection. Solution NMR spectroscopy is ideally suited to the atomic level characterization of intermolecular interfaces and, as a consequence, to epitope discovery. Here, we illustrate how NMR epitope mapping can be used to rapidly and accurately determine protein antigen epitopes. The basic concept is that differences in the NMR signal of an antigen free or bound by an antibody will identify epitope residues. NMR epitope mapping provides more detailed information than mutagenesis or peptide mapping and can be much more rapid than X‐ray crystallography. Advantages and drawbacks of this technique are discussed together with practical considerations. Copyright © 2015 John Wiley & Sons, Ltd.     

Non-mutability by ultraviolet light in uvrD recB derivatives of Escherichia coli WP2 uvrA is due to inhibition of RecA protein activation

The deficiency in UV mutagenesis in uvrD3 recB21 strains of E. coli is almost completely overcome by constitutive activation of RecA protein and expression of the SOS system (by recA730 or 43 degrees C treated recA441 lexA71). When SOS was expressed but RecA protein not self-activated (recA441 lexA71 at 30 degrees C), uvrD3 recB21 still reduced UV mutagenesis at low doses. The uvrD3 recB21 combination is therefore inhibiting activation of RecA protein. It is suggested that the DNA unwinding activity of the products of the uvrD and recB genes may be involved in generating single-stranded DNA needed to activate RecA protein both for the cleavage of LexA repressor and for a further role in UV mutagenesis.     

Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum.     

MAPPING OF HIV-1 GAG EPITOPES RECOGNIZED BY POLYCLONAL ANTIBODIES USING GENE-FRAGMENT PHAGE DISPLAY SYSTEM

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50–250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions.

The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

Mapping of hiv-1 Gag epitopes recognized by polyclonal antibodies using gene-fragment phage display system.

Phage display has emerged as a powerful technique for mapping epitopes recognised by monoclonal and polyclonal antibodies. We have recently developed a simple gene-fragment phage display system and have shown its utility in mapping epitope recognised by a monoclonal antibody. In the present study, we have employed this system in mapping epitopes recognised by polyclonal antibodies raised against HIV-1 capsid protein, p24 which is derived from proteolytic cleavage of Gag polyprotein. HIV-1 gag DNA was fragmented by DNase I and the fragments (50-250 bp) were cloned into gene-fragment phage display vector to construct a library of phages displaying peptides. This phage library was used for affinity selection of phages displaying epitopes recognised by rabbit anti-p24 polyclonal antibodies. Selected phages contained sequences from two discrete regions of p24, demonstrating the presence of two antigenic regions. The DNA sequences encoding these regions were also cloned and expressed as GST fusion proteins. The immunoreactivity of these epitopes as GST fusion proteins, or as phage-displayed peptides, was comparable in ELISA system using same anti-p24 polyclonal antibodies. The results indicate that the gene-fragment based phage display system can be used efficiently to identify epitopes recognised by polyclonal antibodies, and phage displayed epitopes can be directly employed in ELISA to detect antibodies.     

Isolation and characterization of the Vibrio cholerae recA gene

A 3.6-kilobase PstI fragment was isolated from a Vibrio cholerae chromosomal DNA library and shown to encode RecA-like activity in complementation studies with Escherichia coli recA mutants. Although DNA hybridization experiments failed to detect any homology between the E. coli and V. cholerae recA genes, hyperimmune antiserum produced against purified E. coli RecA protein recognized epitopes shared by the V. cholerae protein. The V. cholerae chromosomal fragments, when cloned and transferred to E. coli, provided the missing recA functions, including resistance to the alkylating agent methyl methanesulfonate, resistance to UV irradiation, and promotion of homologous recombination in Hfr mating experiments.     

Analysis of an immunodominant epitope of topoisomerase I in patients with systemic sclerosis

Summary In this paper an immunodominant epitope of Topoisomerase I is described. An epitope expression sublibrary was constructed from Topoisomerase I cDNA. The subclones were screened with an antiserum from a patient with systemic sclerosis (SSc). The positive clones defined one immunodominant B cell epitope (epitope III), which was located at the carboxyterminal part of the protein. The epitope, 52 amino acids in length, neither contains the p 30^gag sequence nor the suggested active site Tyr-723, both presumed antibody recognition sites.More than 70% of our anti-TopoI sera recognize this epitope III, indicating that it is a major recognition site of the anti-TopoI autoantibodies in SSc sera. DNA relaxation experiments show that all sera that recognize epitope III and most sera with antibodies to other epitopes inhibit Topoisomerase I activity.Abbreviations used in this paper SSc Systemic sclerosis - Topol DNA Topoisomerase I (EC 5.99.1.2)     

Characterization of a Novel Dengue Serotype 4 Virus-Specific Neutralizing Epitope on the Envelope Protein Domain III

The dengue virus (DENV) envelope protein domain III (ED3) has been suggested to contain receptor recognition sites and the critical neutralizing epitopes. Up to date, relatively little work has been done on fine mapping of neutralizing epitopes on ED3 for DENV4. In this study, a novel mouse type-specific neutralizing antibody 1G6 against DENV4 was obtained with both prophylactic and therapeutic effects. The epitope was mapped to residues 387–390 of DENV4 envelope protein. Furthermore, site-directed mutagenesis assay identified two critical residues (T388 and H390). The epitope is variable among different DENV serotypes but is highly conserved among four DENV4 genotypes. Affinity measurement showed that naturally occurring variations in ED3 outside the epitope region did not alter the binding of mAb 1G6. These findings expand our understanding of the interactions between neutralizing antibodies and the DENV4 and may be valuable for rational design of DENV vaccines and antiviral drugs.     

鉴定单克隆抗体识别蛋白质抗原表位方法的建立

为了建立鉴定治疗性单克隆抗体识别蛋白质抗原表位的方法,选择程序死亡受体-1 (PD-1) 作为目的蛋白。基于丙氨酸扫描策略,建立了定点突变技术和哺乳动物细胞表达系统相结合的抗原突变体快速表达方法,确定了真核表达元件扩增和细胞转染表达的条件。共表达了150个PD-1蛋白突变体,鉴定了这些突变体与抗PD-1抗体帕博利珠单抗的结合能力。根据蛋白突变体与抗体的结合力并结合蛋白结构分析确定了帕博利珠单抗的抗原表位,与已报道的基于晶体结构的抗原表位高度一致,表明本方法操作简单、准确性高,可用于治疗性单克隆抗体的抗原表位作图。     

Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing

Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day.