DNA polymerase f (Polf) participates in translesion DNA synthesis and is involved in the generation of the majority of mutations induced by DNA damage. The mechanisms that license access of Polf to the primer terminus and regulate the extent of its participation in genome replication are poorly understood. The Polf-dependent damageinduced mutagenesis requires monoubiquitination of proliferating cell nuclear antigen (PCNA) that is triggered by exposure to mutagens. We show that Polf contributes to DNA replication and causes mutagenesis not only in response to DNA damage but also in response to malfunction of normal replicative machinery due to mutations in replication genes. These replication defects lead to ubiquitination of PCNA even in the absence of DNA damage. Unlike damage-induced mutagenesis, the Polf-dependent spontaneous mutagenesis in replication mutants is reduced in strains defective in both ubiquitination and sumoylation of Lys164 of PCNA. Additionally, studies of a PCNA mutant defective for functional interactions with Polf, but not for monoubiquitination by the Rad6/Rad18 complex demonstrate a role for PCNA in regulating the mutagenic activity of Polf separate from its modification at Lys164.

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.

SummaryThe replacement of Escherichia coli recA gene (recA Ec protein from poly(dT); to stabilize a ternary complex RecA::ATP::ssDNA to high salt concentrations; and to be much more rapid in both the nucleation of double-stranded DNA (dsDNA) and the steady-state rate of dsDNA-dependent ATP hydrolysis at pH 7.5. We hypothesized that the high affinity of RecA Pa protein for ssDNA, and especially dsDNA, is the factor that directs the ternary complex to bind secondary DNA to initiate additional acts of recombination instead of to bind LexA repressor to induce constitutive SOS response.

SummaryThe wild-type Escherichia coli RecA protein is a recombinase platform with unrealized recombination potential. We have explored the factors affecting recombination during conjugation with a quantitative assay. Regulatory proteins that affect RecA function have the capacity to increase or decrease recombination frequencies by factors up to sixfold. Autoinhibition by the RecA C-terminus can affect recombination frequency by factors up to fourfold. The greatest changes in recombination frequency measured here are brought about by point mutations in the recA gene. RecA variants can increase recombination frequencies by more than 50-fold. The RecA protein thus possesses an inherently broad functional range. The RecA protein of E. coli (EcRecA) is not optimized for recombination function. Instead, much of the recombination potential of EcRecA is structurally suppressed, probably reflecting cellular requirements. One point mutation in EcRecA with a particularly dramatic effect on recombination frequency, D112R, exhibits an enhanced capacity to load onto SSBcoated ssDNA, overcome the effects of regulatory proteins such as PsiB and RecX, and to pair homologous DNAs. Comparisons of key RecA protein mutants reveal two components to RecA recombination function -filament formation and the inherent DNA pairing activity of the formed filaments.

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.

The ¢lament structures of the self-polymers of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, their complexes with ATPQ QS, phage M13 single-stranded DNA (ssDNA) and the tertiary complexes RecA: :ATPQ QS: :ss-DNA were compared by small angle neutron scattering. A model was developed that allowed for an analytical solution for small angle scattering on a long helical ¢lament, making it possible to obtain the helical pitch and the mean diameter of the protein ¢lament from the scattering curves. The results suggest that the structure of the ¢laments formed by these two RecA proteins, and particularly their complexes with ATPQ QS, is conservative. ß

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65°C (catalytic rate constant of 1.2 to 2.5 min ؊1 at 80 to 95°C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90°C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92°C).RecA protein is a key enzyme of homologous recombination in eubacteria and is also essential to many other aspects of DNA metabolism (for review, see references 3, 11, and 18). To make recombination possible, the RecA protein is polymerized on single-stranded DNA (ssDNA) in the presence of ATP and Mg 2ϩ , forming a helical nucleoprotein filament. This presynaptic recombination complex interacts with double-stranded DNA (dsDNA), aligns homologous sequences between the ssand dsDNA, and promotes the DNA strand exchange by switching DNA pairing within the triple-stranded DNA complex.Although ATP hydrolysis has been shown to not be required for the presynaptic complex formation (10), the latter possesses Mg 2ϩ -and ssDNA-dependent ATPase activity. This activity can easily be used to monitor the RecA protein nucleation as well as saturation of the RecA binding to ssDNA that results in formation of the complex activated for recombination. The in vivo strand exchange process can be modelled in vitro by the transfer of one strand of linear dsDNA to the complementary circular ssDNA (for example, M13 phage); this transfer results in branched DNA recombination intermediates (joint molecules) that are gradually converted into nicked circular heteroduplex dsDNA molecules (final products) and linear ssDNA. As a rule, the circular ssDNA is introduced into the strand exchange reaction in the form of the presynaptic complex. In principle, the strand exchange can proceed in the presence of nonhydrolyzable analogues of ATP, ATP␥S, or ADP-A1F 4 Ϫ (12, 14), but the hydrolysis is required to exchange molecules longer than 1,500 bp as well as to dissociate RecA from the heteroduplex products, to facilitate the bypass of heterologous sequences, and to maintain the reaction polarity (for references, see reference 7).SSB protein is another important component of the strand exchange reaction proceeding under physiological temperature (37°C). This protein affects both the binding of RecA to ssDNA by removing secondary structures from the latter and the completion of strand exchange (2) by covering the linear ssDNA rep...

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