RecF protein targeting to postreplication (daughter strand) gaps I: DNA binding by RecF and RecFR.

In bacteria, the repair of post-replication gaps by homologous recombination requires the action of the recombination mediator proteins RecF, RecO and RecR. Whereas the role of the RecOR proteins to displace the single strand binding protein (SSB) and facilitate RecA loading is clear, how RecF mediates targeting of the system to appropriate sites remains enigmatic. The most prominent hypothesis relies on specific RecF binding to gap ends. To test this idea, we present a detailed examination of RecF and RecFR binding to more than 40 DNA substrates of varying length and structure. Neither RecF nor the RecFR complex exhibited specific DNA binding that can explain the targeting of RecF(R) to post-replication gaps. RecF(R) bound to dsDNA and ssDNA of sufficient length with similar facility. DNA binding was highly ATP-dependent. Most measured Kd values fell into a range of 60-180 nM. The addition of ssDNA extensions on duplex substrates to mimic gap ends or CPD lesions produces only subtle increases or decreases in RecF(R) affinity. Significant RecFR binding cooperativity was evident with many DNA substrates. The results indicate that RecF or RecFR targeting to post-replication gaps must rely on factors not yet identified, perhaps involving interactions with additional proteins.

Interaction with the carboxy-terminal tip of SSB is critical for RecG function in E. coli.

In Escherichia coli, the single-stranded DNA-binding protein (SSB) acts as a genome maintenance organizational hub by interacting with multiple DNA metabolism proteins. Many SSB-interacting proteins (SIPs) form complexes with SSB by docking onto its carboxy-terminal tip (SSB-Ct). An alternative interaction mode in which SIPs bind to PxxP motifs within an intrinsically-disordered linker (IDL) in SSB has been proposed for the RecG DNA helicase and other SIPs. Here, RecG binding to SSB and SSB peptides was measured in vitro and the RecG/SSB interface was identified. The results show that RecG binds directly and specifically to the SSB-Ct, and not the IDL, through an evolutionarily conserved binding site in the RecG helicase domain. Mutations that block RecG binding to SSB sensitize E. coli to DNA damaging agents and induce the SOS DNA-damage response, indicating formation of the RecG/SSB complex is important in vivo. The broader role of the SSB IDL is also investigated. E. coli ssb mutant strains encoding SSB IDL deletion variants lacking all PxxP motifs retain wildtype growth and DNA repair properties, demonstrating that the SSB PxxP motifs are not major contributors to SSB cellular functions.

RecF protein targeting to post-replication (daughter strand) gaps II: RecF interaction with replisomes.

The bacterial RecF, RecO, and RecR proteins are an epistasis group involved in loading RecA protein into post-replication gaps. However, the targeting mechanism that brings these proteins to appropriate gaps is unclear. Here, we propose that targeting may involve a direct interaction between RecF and DnaN. In vivo, RecF is commonly found at the replication fork. Over-expression of RecF, but not RecO or a RecF ATPase mutant, is extremely toxic to cells. We provide evidence that the molecular basis of the toxicity lies in replisome destabilization. RecF over-expression leads to loss of genomic replisomes, increased recombination associated with post-replication gaps, increased plasmid loss, and SOS induction. Using three different methods, we document direct interactions of RecF with the DnaN ß-clamp and DnaG primase that may underlie the replisome effects. In a single-molecule rolling-circle replication system in vitro, physiological levels of RecF protein trigger post-replication gap formation. We suggest that the RecF interactions, particularly with DnaN, reflect a functional link between post-replication gap creation and gap processing by RecA. RecF's varied interactions may begin to explain how the RecFOR system is targeted to rare lesion-containing post-replication gaps, avoiding the potentially deleterious RecA loading onto thousands of other gaps created during replication.

Periplasmic oxidized-protein repair during copper stress in E. coli: A focus on the metallochaperone CusF.

Methionine residues are particularly sensitive to oxidation by reactive oxygen or chlorine species (ROS/RCS), leading to the appearance of methionine sulfoxide in proteins. This post-translational oxidation can be reversed by omnipresent protein repair pathways involving methionine sulfoxide reductases (Msr). In the periplasm of Escherichia coli, the enzymatic system MsrPQ, whose expression is triggered by the RCS, controls the redox status of methionine residues. Here we report that MsrPQ synthesis is also induced by copper stress via the CusSR two-component system, and that MsrPQ plays a role in copper homeostasis by maintaining the activity of the copper efflux pump, CusCFBA. Genetic and biochemical evidence suggest the metallochaperone CusF is the substrate of MsrPQ and our study reveals that CusF methionines are redox sensitive and can be restored by MsrPQ. Thus, the evolution of a CusSR-dependent synthesis of MsrPQ allows conservation of copper homeostasis under aerobic conditions by maintenance of the reduced state of Met residues in copper-trafficking proteins.

HprSR Is a Reactive Chlorine Species-Sensing, Two-Component System in Escherichia coli.

Two-component systems (TCS) are signaling pathways that allow bacterial cells to sense, respond to, and adapt to fluctuating environments. Among the classical TCS of Escherichia coli, HprSR has recently been shown to be involved in the regulation of msrPQ, which encodes the periplasmic methionine sulfoxide reductase system. In this study, we demonstrated that hypochlorous acid (HOCl) induces the expression of msrPQ in an HprSR-dependent manner, whereas H2O2, NO, and paraquat (a superoxide generator) do not. Therefore, HprS appears to be an HOCl-sensing histidine kinase. Using a directed mutagenesis approach, we showed that Met residues located in the periplasmic loop of HprS are important for its activity: we provide evidence that as HOCl preferentially oxidizes Met residues, HprS could be activated via the reversible oxidation of its methionine residues, meaning that MsrPQ plays a role in switching HprSR off. We propose that the activation of HprS by HOCl could occur through a Met redox switch. HprSR appears to be the first characterized TCS able to detect reactive chlorine species (RCS) in E. coli. This study represents an important step toward understanding the mechanisms of RCS resistance in prokaryotes. IMPORTANCE Understanding how bacteria respond to oxidative stress at the molecular level is crucial in the fight against pathogens. HOCl is one of the most potent industrial and physiological microbicidal oxidants. Therefore, bacteria have developed counterstrategies to survive HOCl-induced stress. Over the last decade, important insights into these bacterial protection factors have been obtained. Our work establishes HprSR as a reactive chlorine species-sensing, two-component system in Escherichia coli MG1655, which regulates the expression of msrPQ, two genes encoding, a repair system for HOCl-oxidized proteins. Moreover, we provide evidence suggesting that HOCl could activate HprS through a methionine redox switch.

Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair.

Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

Development of a single-stranded DNA-binding protein fluorescent fusion toolbox.

Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.

Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons.

The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts. Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum.

RecFOR epistasis group: RecF and RecO have distinct localizations and functions in Escherichia coli.

In bacteria, genetic recombination is a major mechanism for DNA repair. The RecF, RecO and RecR proteins are proposed to initiate recombination by loading the RecA recombinase onto DNA. However, the biophysical mechanisms underlying this process remain poorly understood. Here, we used genetics and single-molecule fluorescence microscopy to investigate whether RecF and RecO function together, or separately, in live Escherichia coli cells. We identified conditions in which RecF and RecO functions are genetically separable. Single-molecule imaging revealed key differences in the spatiotemporal behaviours of RecF and RecO. RecF foci frequently colocalize with replisome markers. In response to DNA damage, colocalization increases and RecF dimerizes. The majority of RecF foci are dependent on RecR. Conversely, RecO foci occur infrequently, rarely colocalize with replisomes or RecF and are largely independent of RecR. In response to DNA damage, RecO foci appeared to spatially redistribute, occupying a region close to the cell membrane. These observations indicate that RecF and RecO have distinct functions in the DNA damage response. The observed localization of RecF to the replisome supports the notion that RecF helps to maintain active DNA replication in cells carrying DNA damage.

Spatiotemporal Dynamics of Single-stranded DNA Intermediates in Escherichia coli.

Single-stranded DNA gaps form within the E. coli chromosome during replication, repair and recombination. However, information about the extent of ssDNA creation in the genome is limited. To complement a recent whole-genome sequencing study revealing ssDNA gap genomic distribution, size, and frequency, we used fluorescence microscopy to monitor the spatiotemporal dynamics of single-stranded DNA within live E. coli cells. The ssDNA was marked by a functional fluorescent protein fusion of the SSB protein that replaces the wild type SSB. During log-phase growth the SSB fusion produces a mixture of punctate foci and diffuse fluorescence spread throughout the cytosol. Many foci are clustered. Fluorescent markers of DNA polymerase III frequently co-localize with SSB foci, often localizing to the outer edge of the large SSB features. Novel SSB-enriched features form and resolve regularly during normal growth. UV irradiation induces a rapid increase in SSB foci intensity and produces large features composed of multiple partially overlapping foci. The results provide a critical baseline for further exploration of ssDNA generation during DNA metabolism. Alterations in the patterns seen in a mutant lacking RecB function tentatively suggest associations of particular SSB features with the repair of double strand breaks and post-replication gaps.

Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli.

Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon-insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in ΔpriB cells. Taken together, our results highlight roles for several genes in double-strand DNA break homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli.

Elucidating Recombination Mediator Function Using Biophysical Tools.

The recombination mediator proteins (RMPs) are ubiquitous and play a crucial role in genome stability. RMPs facilitate the loading of recombinases like RecA onto single-stranded (ss) DNA coated by single-strand binding proteins like SSB. Despite sharing a common function, RMPs are the products of a convergent evolution and differ in (1) structure, (2) interaction partners and (3) molecular mechanisms. The RMP function is usually realized by a single protein in bacteriophages and eukaryotes, respectively UvsY or Orf, and RAD52 or BRCA2, while in bacteria three proteins RecF, RecO and RecR act cooperatively to displace SSB and load RecA onto a ssDNA region. Proteins working alongside to the RMPs in homologous recombination and DNA repair notably belongs to the RAD52 epistasis group in eukaryote and the RecF epistasis group in bacteria. Although RMPs have been studied for several decades, molecular mechanisms at the single-cell level are still not fully understood. Here, we summarize the current knowledge acquired on RMPs and review the crucial role of biophysical tools to investigate molecular mechanisms at the single-cell level in the physiological context.

Redox controls RecA protein activity via reversible oxidation of its methionine residues.

Reactive oxygen species (ROS) cause damage to DNA and proteins. Here, we report that the RecA recombinase is itself oxidized by ROS. Genetic and biochemical analyses revealed that oxidation of RecA altered its DNA repair and DNA recombination activities. Mass spectrometry analysis showed that exposure to ROS converted four out of nine Met residues of RecA to methionine sulfoxide. Mimicking oxidation of Met35 by changing it for Gln caused complete loss of function, whereas mimicking oxidation of Met164 resulted in constitutive SOS activation and loss of recombination activity. Yet, all ROS-induced alterations of RecA activity were suppressed by methionine sulfoxide reductases MsrA and MsrB. These findings indicate that under oxidative stress MsrA/B is needed for RecA homeostasis control. The implication is that, besides damaging DNA structure directly, ROS prevent repair of DNA damage by hampering RecA activity.

A variant of the Escherichia coli anaerobic transcription factor FNR exhibiting diminished promoter activation function enhances ionizing radiation resistance.

We have previously generated four replicate populations of ionizing radiation (IR)-resistant Escherichia coli though directed evolution. Sequencing of isolates from these populations revealed that mutations affecting DNA repair (through DNA double-strand break repair and replication restart), ROS amelioration, and cell wall metabolism were prominent. Three mutations involved in DNA repair explained the IR resistance phenotype in one population, and similar DNA repair mutations were prominent in two others. The remaining population, IR-3-20, had no mutations in the key DNA repair proteins, suggesting that it had taken a different evolutionary path to IR resistance. Here, we present evidence that a variant of the anaerobic metabolism transcription factor FNR, unique to and isolated from population IR-3-20, plays a role in IR resistance. The F186I allele of FNR exhibits a diminished ability to activate transcription from FNR-activatable promoters, and furthermore reduces levels of intracellular ROS. The FNR F186I variant is apparently capable of enhancing resistance to IR under chronic irradiation conditions, but does not increase cell survival when exposed to acute irradiation. Our results underline the importance of dose rate on cell survival of IR exposure.

Commercial Lysogeny Broth culture media and oxidative stress: a cautious tale.

Lysogeny Broth (LB), most often misnamed Luria-Bertani medium, ranks among the most commonly used growth media in microbiology. Surprisingly, we observed that oxidative levels vary with the commercial origin of the LB ready to use powder. Indeed, growth on solid media of Escherichia coli and Salmonella derivatives lacking antioxidative stress defenses, such as oxyR mutant devoid of the H2O2-sensing transcriptional activator or Hpx(-) strains lacking catalases and peroxidases, exhibit different phenotypes on LB-Sigma or LB-Difco. Using gene fusion and exogenously added catalase, we found that LB-Sigma contains higher levels of H2O2 than LB-Difco. Also we observed differences in population counts of 82 clinical and environmental isolates of E. coli, depending on the LB used. Further investigations revealed a significant influence of the commercial origin of agar as well. Besides being a warning to the wide population of LB users, our observations provide researchers in the oxidative stress field with a tool to appreciate the severity of mutations in antioxidative stress defenses.

Health literacy weakly but consistently predicts primary care patient dissatisfaction.

OBJECTIVES: To study relationships between health literacy and multiple satisfaction domains. Health literacy is related to some domains of patient satisfaction such as communication and understanding, but little is known about relationships of health literacy with other satisfaction domains. Moreover, the importance of health literacy in predicting satisfaction compared with other patient sociodemographics is underexplored. DESIGN: Cross-sectional survey. SETTING: Primary care waiting areas with a Veterans Administration Medical Center and a university health system. PARTICIPANTS: One thousand five hundred and twenty-eight primary care patients. MAIN OUTCOME MEASURES: A brief demographics questionnaire, the Rapid Estimate of Adult Literacy in Medicine, the Veterans Affairs ambulatory care patient satisfaction survey, and an adaptation of the Charlson Comorbidity Index. RESULTS: In unadjusted regression analyses, lower health literacy level was a significant predictor of worse satisfaction in 7 of 10 domains (P < 0.01). When adjusting for patient sociodemographics, predicted relationships remained significant in six of the seven domains (P < 0.05), with each unit change in the 4-stage literacy classification associated with a 3-6 point decrease in dissatisfaction scores (0-100 scale). Health literacy did not predict overall dissatisfaction (P = 0.55). CONCLUSIONS: These results suggest that health literacy, as assessed through a pronunciation exercise that is closely related to more comprehensive measures of literacy, has a consistent, albeit weak relationship with patient satisfaction. Future work is needed to clarify if patients with lower literacy are really receiving different care than those with higher literacy and to pinpoint the sources of their more negative responses.

Residents as members of intern selection committees: can they partially replace faculty?

BACKGROUND: Faculty involvement in the residency selection process is important, but increasing pressures on available faculty resources have made their ability to participate more difficult. Residents may be acceptable substitutions for some faculty in the selection process. PURPOSE: To test 2 new interview models and explore whether residents could partially replace faculty by examining what role the interview itself plays in the overall assessment of an applicant, comparing faculty and resident ratings of the interview, and asking whether partially substituting residents for faculty had an impact on the match outcome. METHODS: Applicants to an internal medicine residency program were assigned to 1 of 3 interview models: faculty-faculty, resident-faculty, resident-faculty-faculty. The 12 interview days were randomly assigned to a model and all applicants on each day had the same model. Interviewers used an applicant assessment form to assign ratings to 6 components of the portfolio and a final score. RESULTS: For both residents and faculty, the final score was highly correlated with the formal interview component. Within-model analyses showed residents consistently gave more favorable scores than faculty interviewers. There was no impact of interview model on initial or final rank position. Similarly, there was no difference between models in their match "success" rates. CONCLUSIONS: Residents can be successfully substituted for some faculty in the residency selection process. The use of residents does not impact the match results. Developing a small, committed group of interviewers should benefit both programs and applicants.