Peptide-Based Covalent Inhibitors Bearing Mild Electrophiles to Target a Conserved His Residue of the Bacterial Sliding Clamp.

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.

Iterative Structure-Based Optimization of Short Peptides Targeting the Bacterial Sliding Clamp.

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.

Correction: Antibacterial activity of a dual peptide targeting the Escherichia coli sliding clamp and the ribosome.

[This corrects the article DOI: 10.1039/D0CB00060D.].

Proteomic Analysis of DNA Synthesis on a Structured DNA Template in Human Cellular Extracts: Interplay Between NHEJ and Replication-Associated Proteins.

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.

Antibacterial activity of a dual peptide targeting the Escherichia coli sliding clamp and the ribosome.

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.

Interaction of a Model Peptide on Gram Negative and Gram Positive Bacterial Sliding Clamps.

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.

Targeting the replisome with transduced monoclonal antibodies triggers lethal DNA replication stress in cancer cells.

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.

FF483-484 motif of human Polη mediates its interaction with the POLD2 subunit of Polδ and contributes to DNA damage tolerance.

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.

Differential modes of peptide binding onto replicative sliding clamps from various bacterial origins.

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.

Generation of an intrabody-based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells.

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.

Evidence for a Rad18-independent frameshift mutagenesis pathway in human cell-free extracts.

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.

Structure-based design of short peptide ligands binding onto the E. coli processivity ring.

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.

Postreplication repair mechanisms in the presence of DNA adducts in Escherichia 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.

Role of the ubiquitin-binding domain of Polη in Rad18-independent translesion DNA synthesis in human cell extracts.

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.

Distinct beta-clamp interactions govern the activities of the Y family PolIV DNA polymerase.

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.

Kinetics of deoxy-CTP incorporation opposite a dG-C8-N-2-aminofluorene adduct by a high-fidelity DNA polymerase.

The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2'-deoxyguanosine-8-yl)-aminofluorene (G-AF) and the N-2-(2'-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF. We used the DNA polymerase I fragment of a Bacillus stearothermophilus strain as a model for a high-fidelity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF. Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. In the light of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry.

Requirements for PCNA monoubiquitination in human cell-free extracts.

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.

Properties and functions of Escherichia coli: Pol IV and Pol V.

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.

Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative and translesion DNA polymerases.

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 processivity factor beta controls DNA polymerase IV traffic during spontaneous mutagenesis and translesion synthesis in vivo.

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.