Search

The bacterial processivity factor, or sliding clamp (SC), is a target of choice for new antibacterial drugs development. We have previously developed peptides that target Escherichia coli SC and block its interaction with DNA polymerases in vitro. Here, one such SC binding peptide was fused to a Proline-rich AntiMicrobial Peptide (PrAMP) to allow its internalization into E. coli cells. Co-immunoprecipitation assays with a N-terminally modified bifunctional peptide that still enters the bacteria but fails to interact with the bacterial ribosome, the major target of PrAMPs, demonstrate that it actually interacts with the bacterial SC. Moreover, when compared to SC non-binding controls, this peptide induces a ten-fold higher antibacterial activity against E. coli, showing that the observed antimicrobial activity is linked to SC binding. Finally, an unmodified bifunctional compound significantly increases the survival of Drosophila melanogaster flies challenged by an E. coli infection. Our study demonstrates the potential of PrAMPs to transport antibiotics into the bacterial cytoplasm and validates the development of drugs targeting the bacterial processivity factor of Gram-negative bacteria as a promising new class of antibiotics., Bifunctional peptides targeting both the translation and the replication machineries have been developed and shown to act as new antimicrobials.

Bacterial sliding clamps control the access of DNA polymerases to the replication fork and are appealing targets for antibacterial drugs development. It is therefore essential to decipher the polymerase-clamp binding mode across various bacterial species. Here, two residues of the E. coli clamp binding pocket, EcS346 and EcM362, and their cognate residues in M. tuberculosis and B. subtilis clamps, were mutated. The effects of these mutations on the interaction of a model peptide with these variant clamps were evaluated by thermodynamic, molecular dynamics, X-rays crystallography and biochemical analyses. EcM362 and corresponding residues in Gram positive clamps occupy a strategic position where a mobile residue is essential for an efficient peptide interaction. EcS346 has a more subtle function that modulates the pocket folding dynamics, while the equivalent residue in B. subtilis is essential for polymerase activity and might therefore be a Gram positive specific molecular marker. Finally, the peptide binds through an induced-fit process to Gram negative and positive pockets but the complex stability varies according to a pocket specific network of interactions. The following values have no corresponding Zotero field:auth-address: 1 Institut Européen de Chimie et Biologie , Université de Bordeaux-CNRS UMR 5248, CBMN , 2, rue Robert Escarpit , 33607 Pessac , France. 2 Swiss Light Source (SLS) , Paul-Scherrer-Institute (PSI) , 5232 Villigen , Switzerland. 3 Université de Strasbourg , CNRS, Architecture et Réactivité de l'ARN, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS , 15 rue René Descartes , F-67000 Strasbourg , France. 4 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) , Département de Biologie Structurale et Génomique , 1 rue Laurent Fries , BP10142, 67404 Illkirch , France. 5 Biologie et Signalisation Cellulaire, ESBS , UMR7242 CNRS/Université de Strasbourg , Bld S. Brant , 67412 Illkirch Cedex , France.accession-num: 30912430

International audience; The multimeric DNA sliding clamps confer high processivity to replicative DNA polymerases and are also binding platforms for various enzymes involved in DNA metabolism. These enzymes interact with the clamp through a small peptide that binds into a hydrophobic pocket which is a potential target for the development of new antibacterial compounds. Starting from a generic heptapeptide, we used a structure-based strategy to improve the design of new peptide ligands. Chemical modifications at specific residues result in a dramatic increase of the interaction as measured by SPR and ITC. The affinity of our best hits was improved by 2 orders of magnitude as compared to the natural ligand, reaching 10(-8) M range. The molecular basis of the interactions was analyzed by solving the co-crystal structures of the most relevant peptides bound to the clamp and reveals how chemical modifications establish new contacts and contributes to an increased affinity of the ligand.

The genetic information is continuously subjected to the attack by endogenous and exogenous chemical and physical carcinogens that damage the DNA template, thus compromising its biochemical functions. Despite the multiple and efficient DNA repair systems that have evolved to cope with the large variety of damages, some lesions may persist and, as a consequence, interfere with DNA replication. By essence, the damaged-DNA replication process (hereafter termed translesion synthesis or TLS) is a major source of point mutations and is therefore deeply involved in the onset of human diseases such as cancer. Recent identification of numerous DNA polymerases involved in TLS has shed new light onto the molecular mechanisms of mutagenesis. Here, we show that in vivo, both error-free and mutagenic bypass activities of the three DNA polymerases known to be involved in TLS in Escherichia coli (PolII, PolIV and PolV) strictly depend upon the integrity of small peptidic sequences identified as their beta-clamp binding motif. Thus, in addition to its crucial role as the processivity factor of the PolIII replicase, the beta-clamp plays a pivotal role during the TLS process.

Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interact efficiently with our designed peptides and assemble the Escherichia coli and related orthologs clamps, whereas group II clamps poorly interact with the same peptides and include Bacillus subtilis and other Gram-positive clamps. These studies also suggest that the peptide binding process could occur via different mechanisms, which depend on the type of clamp.

The recent discovery of a new family of ubiquitous DNA polymerases involved in translesion synthesis has shed new light onto the biochemical basis of mutagenesis. Among these polymerases, the dinB gene product (Pol IV) is involved in mutagenesis in Escherichia coli. We show here that the activity of native Pol IV is drastically modified upon interaction with the beta subunit, the processivity factor of DNA Pol III. In the absence of the beta subunit Pol IV is strictly distributive and no stable complex between Pol IV and DNA could be detected. In contrast, the beta clamp allows Pol IV to form a stable initiation complex (t 1/2 approximately 2.3 min), which leads to a dramatic increase in the processivity of PoI IV reaching an average of 300-400 nucleotides. In vivo, the beta processivity subunit may target DNA Pol IV to its substrate, generating synthesis tracks much longer than previously thought.

The dinB gene of Escherichia coli is known to be involved in the untargeted mutagenesis of λ phage. Recently, we have demonstrated that this damage-inducible and SOS-controlled gene encodes a novel DNA polymerase, DNA Pol IV, which is able to dramatically increase the untargeted mutagenesis of F′ plasmid. At the amino acid level, DNA Pol IV shares sequence homologies with E. coli UmuC (DNA Pol V), Rev1p, and Rad30p (DNA polymerase η) of Saccharomyces cerevisiae and human Rad30A (XPV) proteins, all of which are involved in translesion DNA synthesis. To better characterize the Pol IV-dependent untargeted mutagenesis, i.e., the DNA Pol IV mutator activity, we analyzed the genetic requirements of this activity and determined the forward mutation spectrum generated by this protein within the c II gene of λ phage. The results indicated that the DNA Pol IV mutator activity is independent of polA , polB , recA , umuDC , uvrA , and mutS functions. The analysis of more than 300 independent mutations obtained in the wild-type or mutS background revealed that the mutator activity clearly promotes single-nucleotide substitutions as well as one-base deletions in the ratio of about 1:2. The base changes were strikingly biased for substitutions toward G:C base pairs, and about 70% of them occurred in 5′-GX-3′ sequences, where X represents the base (T, A, or C) that is mutated to G. These results are discussed with respect to the recently described biochemical characteristics of DNA Pol IV.

By means of specific mutation assays, we show here that in vitro treatment of double-stranded plasmid DNA with methylene blue and white light efficiently promotes frameshift mutagenesis in Escherichia coli. The assays detect either -1 or -2 frameshift mutations within previously characterized hot spot sequences for frameshift mutagenesis induced by the chemical carcinogen N-2-acetylaminofluorene, namely, short runs of contiguous guanines and alternating GpC sequences, respectively. The SOS and umuDC dependences of these mutagenic processes have been investigated. Both -1 and -2 frameshift mutagenesis are increased when the host SOS functions are induced. However, and although functional UmuDC proteins are required for maximal mutation induction, the inducibility of both -1 and -2 frameshift mutagenesis is partially independent upon the integrity of the umuDC operon. In addition, results obtained using plasmids with a site specifically located 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) residue show that this lesion, the major methylene blue plus light induced lesion characterized so far, is inefficient in promoting frameshift mutagenesis. Together, these results led us to conclude that methylene blue plus light treatment of DNA induces, at relatively high rates, lesion(s) other than 8-oxo-dGuo, that efficiently promote(s) frameshift mutagenesis in E. coli.

During bacterial replication, DNA polymerases may encounter DNA lesions that block processive DNA synthesis. Uncoupling the replicative helicase from the stalled DNA polymerase results in the formation of single-stranded DNA (ssDNA) gaps, which are repaired by postreplication repair (PRR), a process that involves at least three mechanisms that collectively remove, circumvent or bypass lesions. RecA mediated excision repair (RAMER) and homologous recombination (HR) are strand-exchange mechanisms that appear to be the predominant strategies for gap repair in the absence of prolonged SOS induction. During RAMER, RecA mediates pairing of damaged ssDNA with an undamaged homologous duplex and subsequent exchange of strands between the damaged and undamaged DNA. Repair of the lesion occurs in the context of the strand-exchange product and is initiated by UvrABC excinuclease; the resulting patch is filled by DNA synthesis using the complementary strand of the homologous duplex as a template. HR uses a complementary strand of an undamaged homologous duplex as a transient template for DNA synthesis. HR requires the formation and resolution of Holliday junctions, and is a mechanism to circumvent the lesion; lesions persisting in one of the daughter DNA duplexes will normally be repaired prior to subsequent rounds of replication/cell division. Translesion DNA Synthesis (TLS) does not involve strand-exchange mechanisms; it is carried out by specialized DNA polymerases that are able to catalyze nucleotide incorporation opposite lesions that cannot be bypassed by high-fidelity replicative polymerases. Maximum levels of TLS occur during prolonged SOS induction generally associated with increased mutagenesis. RAMER, HR and TLS are alternative mechanisms for processing a common intermediate-the ssDNA gap containing a RecA nucleofilament. The actual pathway that is utilized will be strongly influenced by multiple factors, including the blocking/coding capacity of the lesion, the nature of the gene products that can be assembled at the ssDNA gap, the availability of a homologous partner for RAMER and HR, and protein:protein interactions and post-translational modifications that modulate the mutagenic activity of Pol-IV and Pol-V.

Most DNA polymerases interact with their cognate processive replication factor through a small peptide, this interaction being absolutely required for their function in vivo. We have solved the crystal structure of a complex between the beta sliding clamp of Escherichia coli and the 16 residue C-terminal peptide of Pol IV (P16). The seven C-terminal residues bind to a pocket located at the surface of one beta monomer. This region was previously identified as the binding site of another beta clamp binding protein, the delta subunit of the gamma complex. We show that peptide P16 competitively prevents beta-clamp-mediated stimulation of both Pol IV and alpha subunit DNA polymerase activities, suggesting that the site of interaction of the alpha subunit with beta is identical with, or overlaps that of Pol IV. This common binding site for delta, Pol IV and alpha subunit is shown to be formed by residues that are highly conserved among many bacterial beta homologs, thus defining an evolutionarily conserved hydrophobic crevice for sliding clamp ligands and a new target for antibiotic drug design.

The biochemistry and genetics of translesion synthesis (TLS) and, as a consequence, of mutagenesis has recently received much attention in view of the discovery of novel DNA polymerases, most of which belong to the Y family. These distributive and low fidelity enzymes assist the progression of the high fidelity replication complex in the bypass of DNA lesions that normally hinder its progression. The present paper extends our previous observation that in Escherichia coli all three SOS-inducible DNA polymerases (Pol II, IV and V) are involved in TLS and mutagenesis [1]. The genetic control of frameshift mutation pathways induced by N-2-acetylaminofluorene (AAF) adducts or by oxidative lesions induced by methylene blue and visible light is investigated. The data show various examples of mutation pathways with an absolute requirement for a specific combination of DNA polymerases and, in contrast, other examples where two DNA polymerases exhibit functional redundancy within the same pathway. We suggest that cells respond to the challenge of replicating DNA templates potentially containing a large diversity of DNA lesions by using a pool of accessory DNA polymerases with relaxed specificities that assist the high fidelity replicase.

The dinB-encoded DNA polymerase IV (Pol IV) belongs to the recently identified Y-family of DNA polymerases. Like other members of this family, Pol IV is involved in translesion synthesis and mutagenesis. Here, we show that the C-terminal five amino acids of Pol IV are essential in targeting it to the beta-clamp, the processivity factor of the replicative DNA polymerase (Pol III) of Escherichia coli. In vivo, the disruption of this interaction obliterates the function of Pol IV in both spontaneous and induced mutagenesis. These results point to the pivotal role of the processivity clamp during DNA polymerase trafficking in the vicinity of damaged-template DNA.

Most organisms contain several members of a recently discovered class of DNA polymerases (umuC/dinB superfamily) potentially involved in replication of damaged DNA. In Escherichia coli, only Pol V (umuDC) was known to be essential for base substitution mutagenesis induced by UV light or abasic sites. Here we show that, depending upon the nature of the DNA damage and its sequence context, the two additional SOS-inducible DNA polymerases, Pol II (polB) and Pol IV (dinB), are also involved in error-free and mutagenic translesion synthesis (TLS). For example, bypass of N:-2-acetylaminofluorene (AAF) guanine adducts located within the NAR:I mutation hot spot requires Pol II for -2 frameshifts but Pol V for error-free TLS. On the other hand, error-free and -1 frameshift TLS at a benzo(a)pyrene adduct requires both Pol IV and Pol V. Therefore, in response to the vast diversity of existing DNA damage, the cell uses a pool of 'translesional' DNA polymerases in order to bypass the various DNA lesions.

In Escherichia coli , the dinB gene is required for the SOS-induced λ untargeted mutagenesis pathway and confers a mutator phenotype to the cell when the gene product is overexpressed. Here, we report that the purified DinB protein is a DNA polymerase. This novel E. coli DNA polymerase (pol IV) is shown to be strictly distributive, devoid of proofreading activity, and prone to elongate bulged (misaligned) primer/template structures. Site-directed mutagenesis experiments of dinB also demonstrate that the polymerase activity of DinB is required for its in vivo mutagenicity. Along with the sequence homologies previously found within the UmuC-like protein family, these results indicate that the uncovered DNA polymerase activity may be a common feature of all these homologous proteins.

We have previously shown that a single N-2-acetylaminofluorene (AAF) adduct bound to the C-8 position of a guanine residue located within plasmids containing the unidirectional ColE1 origin of replication induces a 20-fold higher mutation frequency when the adduct is located in the lagging strand as compared to the leading strand. In this study, single 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) lesions have been intro duced in the leading and lagging strand orientation within the same sequence context as for the AAF adducts. The induced frequency of guanine to thymine transversions has been measured, using a specific PCR-based quantitative assay, in strains deficient in the repair of the oxidative lesion. The potential involvement of the UvrABC excision repair system in the removal of 8-oxodG has also been investigated and ruled out. Concerning the mutation frequency asymmetry, in contrast to AAF adducts, 8-oxodG adducts induce the same mutation frequency, irrespective of their location in the leading or lagging strands. This striking difference between 8-oxodG and dGuo-C8-AAF adducts is discussed in terms of their differential capacity to block DNA replication.