Protealysin Targets the Bacterial Housekeeping Proteins FtsZ and RecA.

Serratia proteamaculans synthesizes the intracellular metalloprotease protealysin. This work was aimed at searching for bacterial substrates of protealysin among the proteins responsible for replication and cell division. We have shown that protealysin unlimitedly cleaves the SOS response protein RecA. Even 20% of the cleaved RecA in solution appears to be incorporated into the polymer of uncleaved monomers, preventing further polymerization and inhibiting RecA ATPase activity. Transformation of Escherichia coli with a plasmid carrying the protealysin gene reduces the bacterial UV survival up to 10 times. In addition, the protealysin substrate is the FtsZ division protein, found in both E. coli and Acholeplasma laidlawii, which is only 51% identical to E. coli FtsZ. Protealysin cleaves FtsZ at the linker between the globular filament-forming domain and the C-terminal peptide that binds proteins on the bacterial membrane. Thus, cleavage of the C-terminal segment by protealysin can lead to the disruption of FtsZ's attachment to the membrane, and thereby inhibit bacterial division. Since the protealysin operon encodes not only the protease, but also its inhibitor, which is typical for the system of interbacterial competition, we assume that in the case of penetration of protealysin into neighboring bacteria that do not synthesize a protealysin inhibitor, cleavage of FtsZ and RecA by protealysin may give S. proteamaculans an advantage in interbacterial competition.

A new insight into RecA filament regulation by RecX from the analysis of conformation-specific interactions.

RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on ssDNA in an ATP-dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (Escherichia coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.

Targeting evolution of antibiotic resistance by SOS response inhibition.

Antibiotic resistance is acquired in response to antibiotic therapy by activating SOS-depended mutagenesis and horizontal gene transfer pathways. Compounds able to inhibit SOS response are extremely important to develop new combinatorial strategies aimed to block mutagenesis. The regulators of homologous recombination involved in the processes of DNA repair should be considered as potential targets for blocking. This review highlights the current knowledge of the protein targets for the evolution of antibiotic resistance and the inhibitory effects of some new compounds on this pathway.

Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA.

Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

Single-molecule analysis reveals two distinct states of the compressed RecA filament on single-stranded DNA.

The RecA protein plays a key role in bacterial homologous recombination (HR) and acts through assembly of long helical filaments around single-stranded DNA in the presence of ATP. Large-scale conformational changes induced by ATP hydrolysis result in transitions between stretched and compressed forms of the filament. Here, using a single-molecule approach, we show that compressed RecA nucleoprotein filaments can exist in two distinct interconvertible states depending on the presence of ADP in the monomer-monomer interface. Binding of ADP promotes cooperative conformational transitions and directly affects mechanical properties of the filament. Our findings reveal that RecA nucleoprotein filaments are able to continuously cycle between three mechanically distinct states that might have important implications for RecA-mediated processes of HR.

Systematic search for structural motifs of peptide binding to double-stranded DNA.

A large variety of short biologically active peptides possesses antioxidant, antibacterial, antitumour, anti-ageing and anti-inflammatory activity, involved in the regulation of neuro-immuno-endocrine system functions, cell apoptosis, proliferation and differentiation. Therefore, the mechanisms of their biological activity are attracting increasing attention not only in modern molecular biology, biochemistry and biophysics, but also in pharmacology and medicine. In this work, we systematically analysed the ability of dipeptides (all possible combinations of the 20 standard amino acids) to bind all possible combinations of tetra-nucleotides in the central part of dsDNA in the classic B-form using molecular docking and molecular dynamics. The vast majority of the dipeptides were found to be unable to bind dsDNA. However, we were able to identify 57 low-energy dipeptide complexes with peptide-dsDNA possessing high selectivity for DNA binding. The analysis of the dsDNA complexes with dipeptides with free and blocked N- and C-terminus showed that selective peptide binding to dsDNA can increase dramatically with the peptide length.

Blocking the RecA activity and SOS-response in bacteria with a short α-helical peptide.

The RecX protein, a very active natural RecA protein inhibitor, can completely disassemble RecA filaments at nanomolar concentrations that are two to three orders of magnitude lower than that of RecA protein. Based on the structure of RecX protein complex with the presynaptic RecA filament, we designed a short first in class α-helical peptide that both inhibits RecA protein activities in vitro and blocks the bacterial SOS-response in vivo. The peptide was designed using SEQOPT, a novel method for global sequence optimization of protein α-helices. SEQOPT produces artificial peptide sequences containing only 20 natural amino acids with the maximum possible conformational stability at a given pH, ionic strength, temperature, peptide solubility. It also accounts for restrictions due to known amino acid residues involved in stabilization of protein complexes under consideration. The results indicate that a few key intermolecular interactions inside the RecA protein presynaptic complex are enough to reproduce the main features of the RecX protein mechanism of action. Since the SOS-response provides a major mechanism of bacterial adaptation to antibiotics, these results open new ways for the development of antibiotic co-therapy that would not cause bacterial resistance.

DNA Metabolism in Balance: Rapid Loss of a RecA-Based Hyperrec Phenotype.

The RecA recombinase of Escherichia coli has not evolved to optimally promote DNA pairing and strand exchange, the key processes of recombinational DNA repair. Instead, the recombinase function of RecA protein represents an evolutionary compromise between necessary levels of recombinational DNA repair and the potentially deleterious consequences of RecA functionality. A RecA variant, RecA D112R, promotes conjugational recombination at substantially enhanced levels. However, expression of the D112R RecA protein in E. coli results in a reduction in cell growth rates. This report documents the consequences of the substantial selective pressure associated with the RecA-mediated hyperrec phenotype. With continuous growth, the deleterious effects of RecA D112R, along with the observed enhancements in conjugational recombination, are lost over the course of 70 cell generations. The suppression reflects a decline in RecA D112R expression, associated primarily with a deletion in the gene promoter or chromosomal mutations that decrease plasmid copy number. The deleterious effects of RecA D112R on cell growth can also be negated by over-expression of the RecX protein from Neisseria gonorrhoeae. The effects of the RecX proteins in vivo parallel the effects of the same proteins on RecA D112R filaments in vitro. The results indicate that the toxicity of RecA D112R is due to its persistent binding to duplex genomic DNA, creating barriers for other processes in DNA metabolism. A substantial selective pressure is generated to suppress the resulting barrier to growth.

Structure of RecX protein complex with the presynaptic RecA filament: Molecular dynamics simulations and small angle neutron scattering.

Using molecular modeling techniques we have built the full atomic structure and performed molecular dynamics simulations for the complexes formed by Escherichia coli RecX protein with a single-stranded oligonucleotide and with RecA presynaptic filament. Based on the modeling and SANS experimental data a sandwich-like filament structure formed two chains of RecX monomers bound to the opposite sides of the single stranded DNA is proposed for RecX::ssDNA complex. The model for RecX::RecA::ssDNA include RecX binding into the grove of RecA::ssDNA filament that occurs mainly via Coulomb interactions between RecX and ssDNA. Formation of RecX::RecA::ssDNA filaments in solution was confirmed by SANS measurements which were in agreement with the spectra computed from the molecular dynamics simulations.

SSB antagonizes RecX-RecA interaction.

The RecX protein of Escherichia coli inhibits the extension of RecA protein filaments on DNA, presumably by binding to and blocking the growing filament end. The direct binding of RecX protein to single-stranded DNA is weak, and previous reports suggested that direct binding to DNA did not explain the effects of RecX. We now demonstrate that elevated concentrations of SSB greatly moderate the effects of RecX protein. High concentrations of the yeast RPA protein have the same effect, suggesting that the effect is not species-specific or even specific to bacterial SSB proteins. A direct SSB-RecX interaction is thus unlikely. We suggest that SSB is blocking access to single-stranded DNA. The evident competition between RecX and SSB implies that the mechanism of RecX action may involve RecX binding to both RecA protein and to DNA. We speculate that the interaction of RecX protein and RecA may enable an enhanced DNA binding by RecX protein. The effects of SSB are increased if the SSB C terminus is removed.

Two RecA protein types that mediate different modes of hyperrecombination.

RecAX53 is a chimeric variant of the Escherichia coli RecA protein (RecAEc) that contains a part of the central domain of Pseudomonas aeruginosa RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in E. coli cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the mutS gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers.

Distinguishing characteristics of hyperrecombinogenic RecA protein from Pseudomonas aeruginosa acting in Escherichia coli.

In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecA(Pa)) exhibits a more robust recombinase activity than its E. coli counterpart (RecA(Ec)). Low-level expression of RecA(Pa) in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecA(Ec). This genetic effect is supported by the biochemical finding that the RecA(Pa) protein is more efficient in filament formation than RecA K72R, a mutant protein with RecA(Ec)-like DNA-binding ability. Expression of RecA(Pa) also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecA(Pa) filaments initiate recombination equally from both the 5' and 3' ends. Besides, these filaments exhibit more resistance to disassembly from the 5' ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities.

Hyper-recombinogenic RecA protein from Pseudomonas aeruginosa with enhanced activity of its primary DNA binding site.

According to one prominent model, each protomer in the activated nucleoprotein filament of homologous recombinase RecA possesses two DNA-binding sites. The primary site binds (1) single-stranded DNA (ssDNA) to form presynaptic complex and (2) the newly formed double-stranded (ds) DNA whereas the secondary site binds (1) dsDNA of a partner to initiate strand exchange and (2) the displaced ssDNA following the strand exchange. RecA protein from Pseudomonas aeruginosa (RecAPa) promotes in Escherichia coli hyper-recombination in an SOS-independent manner. Earlier we revealed that RecAPa rapidly displaces E.coli SSB protein (SSB-Ec) from ssDNA to form presynaptic complex. Here we show that this property (1) is based on increased affinity of ssDNA for the RecAPa primary DNA binding site while the affinity for the secondary site remains similar to that for E.coli RecA, (2) is not specific for SSB-Ec but is also observed for SSB protein from P.aeruginosa that, in turn, predicts a possibility of enhanced recombination repair in this pathogenic bacterium.