RESUMEN
It is established that short inverted repeats trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with structured DNA is unknown. It has been previously reported that in human cell-free extracts, DNA primer extension using a structured single-stranded template is transiently blocked at DNA hairpins. Here, the proteomic analysis of proteins bound to the DNA template is reported and evidence that the DNA-PK complex (DNA-PKcs and the Ku heterodimer) recognizes, and is activated by, structured single-stranded DNA is provided. Hijacking the DNA-PK complex by double-stranded oligonucleotides results in a large removal of the pausing sites and an elevated DNA extension efficiency. Conversely, DNA-PKcs inhibition results in its stabilization on the template, along with other proteins acting downstream in the Non-Homologous End-Joining (NHEJ) pathway, especially the XRCC4-DNA ligase 4 complex and the cofactor PAXX. Retention of NHEJ factors to the DNA in the absence of DNA-PKcs activity correlates with additional halts of primer extension, suggesting that these proteins hinder the progression of the DNA synthesis at these sites. Overall these results raise the possibility that, upon binding to hairpins formed onto ssDNA during fork progression, the DNA-PK complex interferes with replication fork dynamics in vivo.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Replicación del ADN , ADN/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Extractos Celulares , ADN/química , ADN/genética , ADN Ligasa (ATP)/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Autoantígeno Ku/metabolismo , Proteínas Nucleares/metabolismo , Unión ProteicaRESUMEN
Although chemical inhibition of the DNA damage response (DDR) in cancer cells triggers cell death, it is not clear if the fork blockade achieved with inhibitors that neutralise proteins of the replisome is sufficient on its own to overcome the DDR. Monoclonal antibodies to PCNA, which block the DNA elongation process in vitro, have been developed. When these antibodies were transduced into cancer cells, they are able to inhibit the incorporation of nucleoside analogues. When co-delivered with anti-PCNA siRNA, the cells were flattened and the size of their nuclei increased by up to 3-fold, prior to cell death. Analysis of these nuclei by super-resolution microscopy revealed the presence of large numbers of phosphorylated histone H2AX foci. A senescence-like phenotype of the transduced cells was also observed upon delivery of the corresponding Fab molecules or following PCNA gene disruption or when the Fab fragment of an antibody that neutralises DNA polymerase alpha was used. Primary melanoma cells and leukaemia cells that are resistant to chemical inhibitors were similarly affected by these antibody treatments. These results demonstrate that transduced antibodies can trigger a lethal DNA replication stress, which kills cancer cells by abolishing the biological activity of several constituents of the replisome.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/farmacología , Antineoplásicos/farmacología , Replicación del ADN/efectos de los fármacos , ADN de Neoplasias/genética , Animales , Roturas del ADN de Doble Cadena , ADN Polimerasa III/antagonistas & inhibidores , ADN de Neoplasias/metabolismo , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Técnicas de Silenciamiento del Gen , Células HeLa , Histonas/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Ratones Endogámicos BALB C , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/inmunología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Estrés FisiológicoRESUMEN
Switching between replicative and translesion synthesis (TLS) DNA polymerases are crucial events for the completion of genomic DNA synthesis when the replication machinery encounters lesions in the DNA template. In eukaryotes, the translesional DNA polymerase η (Polη) plays a central role for accurate bypass of cyclobutane pyrimidine dimers, the predominant DNA lesions induced by ultraviolet irradiation. Polη deficiency is responsible for a variant form of the Xeroderma pigmentosum (XPV) syndrome, characterized by a predisposition to skin cancer. Here, we show that the FF483-484 amino acids in the human Polη (designated F1 motif) are necessary for the interaction of this TLS polymerase with POLD2, the B subunit of the replicative DNA polymerase δ, both in vitro and in vivo. Mutating this motif impairs Polη function in the bypass of both an N-2-acetylaminofluorene adduct and a TT-CPD lesion in cellular extracts. By complementing XPV cells with different forms of Polη, we show that the F1 motif contributes to the progression of DNA synthesis and to the cell survival after UV irradiation. We propose that the integrity of the F1 motif of Polη, necessary for the Polη/POLD2 interaction, is required for the establishment of an efficient TLS complex.
Asunto(s)
Daño del ADN , ADN Polimerasa III/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Ciclo Celular , Línea Celular , Supervivencia Celular , Humanos , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/metabolismo , Rayos UltravioletaRESUMEN
Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single-chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti-PCNA scFvs were screened in vitro after being tagged with dimeric glutathione-S-transferase. Anti-PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error-prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C-terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide-conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody-based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy.
Asunto(s)
Diagnóstico por Imagen , Neoplasias/diagnóstico , Antígeno Nuclear de Célula en Proliferación/metabolismo , Secuencia de Aminoácidos , Afinidad de Anticuerpos , Línea Celular Tumoral , Supervivencia Celular , Fluorescencia , Humanos , Indicadores y Reactivos , Datos de Secuencia Molecular , Biblioteca de Péptidos , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunología , Fracciones Subcelulares/metabolismoRESUMEN
Peptide-based covalent inhibitors targeted to nucleophilic protein residues have recently emerged as new modalities to target protein-protein interactions (PPIs) as they may provide some benefits over more classic competitive inhibitors. Covalent inhibitors are generally targeted to cysteine, the most intrinsically reactive amino acid residue, and to lysine, which is more abundant at the surface of proteins but much less frequently to histidine. Herein, we report the structure-guided design of targeted covalent inhibitors (TCIs) able to bind covalently and selectively to the bacterial sliding clamp (SC), by reacting with a well-conserved histidine residue located on the edge of the peptide-binding pocket. SC is an essential component of the bacterial DNA replication machinery, identified as a promising target for the development of new antibacterial compounds. Thermodynamic and kinetic analyses of ligands bearing different mild electrophilic warheads confirmed the higher efficiency of the chloroacetamide compared to Michael acceptors. Two high-resolution X-ray structures of covalent inhibitor-SC adducts were obtained, revealing the canonical orientation of the ligand and details of covalent bond formation with histidine. Proteomic studies were consistent with a selective SC engagement by the chloroacetamide-based TCI. Finally, the TCI of SC was substantially more active than the parent noncovalent inhibitor in an in vitro SC-dependent DNA synthesis assay, validating the potential of the approach to design covalent inhibitors of protein-protein interactions targeted to histidine.
RESUMEN
In eukaryotic cells, the Rad6/Rad18-dependent monoubiquitination of the proliferating cell nuclear antigen (PCNA) plays an essential role in the switching between replication and translesion DNA synthesis (TLS). The DNA polymerase Polη binds to PCNA via a consensus C-terminal PCNA-interacting protein (PIP) motif. It also specifically interacts with monoubiquitinated PCNA thanks to a recently identified ubiquitin-binding domain (UBZ). To investigate whether the TLS activity of Polη is always coupled to PCNA monoubiquitination, we monitor the ability of cell-free extracts to perform DNA synthesis across different types of lesions. We observe that a cis-syn cyclobutane thymine dimer (TT-CPD), but not a N-2-acetylaminofluorene-guanine (G-AAF) adduct, is efficiently bypassed in extracts from Rad18-deficient cells, thus demonstrating the existence of a Polη-dependent and Rad18-independent TLS pathway. In addition, by complementing Polη-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Polη activity. The finding that the bypass of a CPD lesion in vitro does not require Ub-PCNA but nevertheless depends on the UBZ domain of Polη, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/fisiología , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , Extractos Celulares , Línea Celular , Supervivencia Celular/efectos de la radiación , Aductos de ADN/química , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Técnicas de Inactivación de Genes , Humanos , Mutación , Estructura Terciaria de Proteína , Dímeros de Pirimidina/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Aductos de ADN , Reparación del ADN , Escherichia coli/genética , Replicación del ADN , ADN Bacteriano/genética , ADN Cruciforme/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Modelos Biológicos , Procesamiento Proteico-Postraduccional , Respuesta SOS en GenéticaRESUMEN
The bacterial DNA sliding clamp (SC), or replication processivity factor, is a promising target for the development of novel antibiotics. We report a structure-activity relationship study of a new series of peptides interacting within the Escherichia coli SC (EcSC) binding pocket. Various modifications were explored including N-alkylation of the peptide bonds, extension of the N-terminal moiety, and introduction of hydrophobic and constrained residues at the C-terminus. In each category, single modifications were identified that increased affinity to EcSC. A combination of such modifications yielded in several cases to a substantially increased affinity compared to the parent peptides with Kd in the range of 30-80 nM. X-ray structure analysis of 11 peptide/EcSC co-crystals revealed new interactions at the peptide-protein interface (i.e., stacking interactions, hydrogen bonds, and hydrophobic contacts) that can account for the improved binding. Several compounds among the best binders were also found to be more effective in inhibiting SC-dependent DNA synthesis.
Asunto(s)
Escherichia coli/química , Péptidos/química , Cristalización , Cristalografía por Rayos X , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Conformación Proteica , Relación Estructura-Actividad , TermodinámicaRESUMEN
[This corrects the article DOI: 10.1039/D0CB00060D.].
RESUMEN
The prototypic Y family DNA polymerase IV (PolIV) of Escherichia coli is involved in multiple replication-associated processes including spontaneous mutagenesis, translesion synthesis (TLS), cell fitness, survival under stressful conditions and checkpoint like functions. It interacts physically and functionally with the replisome's beta processivity clamp through the canonical PolIV C-terminal peptide (CTP). A second interaction that involves a portion of the little finger (LF) domain of PolIV has been structurally described. Here we show that the LF-beta interaction stabilizes the clamp-polymerase complex in vitro and is necessary for the access of PolIV to ongoing replication forks in vivo. However, in contrast to the CTP-beta, the LF-beta interaction is dispensable for the role of the polymerase in TLS. This discloses two independent modes of action for PolIV and, in turn, uncovers a novel way by which the cell may regulate the potentially deleterious effect of such low fidelity polymerases during replication.
Asunto(s)
ADN Polimerasa III/metabolismo , ADN Polimerasa beta/metabolismo , Replicación del ADN , Escherichia coli/metabolismo , ADN Polimerasa beta/genética , Reparación del ADN , ADN Bacteriano/genética , Escherichia coli/genética , Modelos Moleculares , Mutagénesis , Mutación , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de ProteínasRESUMEN
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.
RESUMEN
Bacterial sliding clamps control the access of DNA polymerases to the replication fork and are appealing targets for antibacterial drug 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.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Péptidos/farmacología , Cristalografía por Rayos X , ADN Polimerasa Dirigida por ADN/metabolismo , Desarrollo de Medicamentos , Escherichia coli/genética , Bacterias Grampositivas/genética , Ligandos , Modelos Moleculares , Mutación , Inhibidores de la Síntesis del Ácido Nucleico , Péptidos/química , Unión Proteica , Conformación ProteicaRESUMEN
The Rad6/Rad18-dependent monoubiquitination of PCNA plays a crucial role in regulating replication past DNA damage in eukaryotic cells. We show here that in human cell-free extracts, efficient PCNA monoubiquitination requires both the synthesis of relatively long DNA tracts and polymerase idling or stalling at sites of DNA modification or DNA secondary structures. This dual dependency suggests a dynamic process in which, following initiation, the DNA synthesizing complex undergoes modifications that make it competent as a mediator for the activation of the Rad6/Rad18 pathway.
Asunto(s)
Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina/metabolismo , Sistema Libre de Células , Replicación del ADN , Células HeLa , HumanosRESUMEN
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. 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.
Asunto(s)
ADN Polimerasa II/fisiología , ADN Polimerasa beta/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , Escherichia coli/enzimología , Escherichia coli/genética , Mutagénesis/genética , 2-Acetilaminofluoreno/toxicidad , Carcinógenos/toxicidad , Aductos de ADN , Daño del ADN/efectos de los fármacos , Replicación del ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli , Mutación del Sistema de Lectura , Secuencia Rica en GC , Luz , Azul de Metileno/farmacología , Mutagénesis/efectos de la radiación , Oxidación-Reducción , Respuesta SOS en GenéticaRESUMEN
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.
Asunto(s)
ADN Polimerasa II/fisiología , ADN Polimerasa beta/fisiología , Reparación del ADN , ADN Bacteriano/biosíntesis , ADN Polimerasa Dirigida por ADN/fisiología , Escherichia coli/enzimología , Mutagénesis , Mutación , Daño del ADN , Replicación del ADN/fisiología , Escherichia coli/genética , Proteínas de Escherichia coli , Subunidades de ProteínaRESUMEN
Escherichia coli possesses two members of the newly discovered class of Y DNA polymerases (Ohmori et al., 2001): Pol IV (dinB) and Pol V (umuD'C). Polymerases that belong to this family are often referred to as specialized or error-prone DNA polymerases to distinguish them from the previously discovered DNA polymerases (Pol I, II, and III) that are essentially involved in DNA replication or error-free DNA repair. Y-family DNA polymerases are characterized by their capacity to replicate DNA, through chemically damaged template bases, or to elongate mismatched primer termini. These properties stem from their capacity to accommodate and use distorted primer templates within their active site and from the lack of an associated exonuclease activity. Even though both belong to the Y-family, Pol IV and Pol V appear to perform distinct physiological functions. Although Pol V is clearly the major lesion bypass polymerase involved in damage-induced mutagenesis, the role of Pol IV remains enigmatic. Indeed, compared to a wild-type strain, a dinB mutant exhibits no clear phenotype with respect to survival or mutagenesis following treatment with DNA-damaging agents. Subtler dinB phenotypes will be discussed below. Moreover, despite the fact that both dinB and umuDC loci are controlled by the SOS response, their constitutive and induced levels of expression are dramatically different. In noninduced cells, Pol V is undetectable by Western analysis. In contrast, it is estimated that there are about 250 copies of Pol IV per cell. On SOS induction, it is believed that only about 15 molecules of Pol V are assembled per cell (S. Sommer, personal communication), whereas Pol IV levels reach approximately 2500 molecules. In fact, despite extensive knowledge of the individual enzymatic properties of all five E. coli DNA polymerases, much more work is needed to understand how their activities are orchestrated within a living cell.
Asunto(s)
ADN Polimerasa beta/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/enzimología , ADN Polimerasa beta/química , Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , Proteínas de Escherichia coliRESUMEN
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.
Asunto(s)
Unión Competitiva , ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Replicación del ADN/genética , Escherichia coli/enzimología , Fragmentos de Péptidos/metabolismo , Cristalización , ADN Polimerasa I/metabolismo , ADN Polimerasa III/metabolismo , ADN Polimerasa beta/genética , ADN Bacteriano/genética , Cinética , Ligandos , Modelos Moleculares , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Replicación del ADN , Péptidos/metabolismo , Secuencia de Aminoácidos , Antibacterianos/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Alineación de Secuencia , TermodinámicaRESUMEN
Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF)-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G) and 5'-GGCGCC-3' (NarI site), induces -1 and -2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion.
Asunto(s)
Extractos Celulares/genética , Replicación del ADN/genética , Mutación del Sistema de Lectura/genética , Mutagénesis/genética , 2-Acetilaminofluoreno , Animales , Células COS , Sistema Libre de Células , Chlorocebus aethiops , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Guanina/metabolismo , Células HCT116 , Humanos , Mutación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
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.