Controlling genome topology with sequences that trigger post-replication gap formation during replisome passage: the E. coli RRS elements.

We report that the Escherichia coli chromosome includes novel GC-rich genomic structural elements that trigger formation of post-replication gaps upon replisome passage. The two nearly perfect 222 bp repeats, designated Replication Risk Sequences or RRS, are each 650 kb from the terminus sequence dif and flank the Ter macrodomain. RRS sequence and positioning is highly conserved in enterobacteria. At least one RRS appears to be essential unless a 200 kb region encompassing one of them is amplified. The RRS contain a G-quadruplex on the lagging strand which impedes DNA polymerase extension producing lagging strand ssDNA gaps, $ \le$2000 bp long, upon replisome passage. Deletion of both RRS elements has substantial effects on global genome structure and topology. We hypothesize that RRS elements serve as topological relief valves during chromosome replication and segregation. There have been no screens for genomic sequences that trigger transient gap formation. Functional analogs of RRS could be widespread, possibly including some enigmatic G-quadruplexes in eukaryotes.

Towards personalised anti-microbial and immune approaches to infections in acute care. Can real-time genomic-informed diagnosis of pathogens, and immune-focused therapies improve outcomes for patients? An observational, experimental study protocol.

INTRODUCTION: Infection causes a vast burden of disease, with significant mortality, morbidity and costs to health-care systems. However, identifying the pathogen causative infection can be challenging, resulting in high use of broad-spectrum antibiotics, much of which may be inappropriate. Novel metagenomic methods have potential to rapidly identify pathogens, however their clinical utility for many infections is currently unclear. Outcome from infection is also impacted by the effectiveness of immune responses, which can be impaired by age, co-morbidity and the infection itself. The aims of this study are twofold: To compare diversity of organisms identified and time-to-result using metagenomic methods versus traditional culture -based techniques, to explore the potential clinical role of metagenomic approaches to pathogen identification in a range of infections.To characterise the ex vivo function of immune cells from patients with acute infection, exploring host and pathogen-specific factors which may affect immune function and overall outcomes. METHODS: This is a prospective observational study of patients with acute infection. Patients with symptoms suggestive of an acute infection will be recruited, and blood and bodily fluid relevant to the site of infection collected (for example, sputum and naso-oropharyngeal swabs for respiratory tract infections, or urine for a suspected urinary tract infection). Metagenomic analysis of samples will be compared to traditional microbiology, alongside the antimicrobials received. Blood and respiratory samples such as bronchoalveolar lavage will be used to isolate immune cells and interrogate immune cell function. Where possible, similar samples will be collected from matched participants without a suspected infection to determine the impact of infection on both microbiome and immune cell function.

The intrinsically disordered linker in the single-stranded DNA-binding protein influences DNA replication restart and recombination pathways in Escherichia coli K-12.

Tetrameric single-stranded (ss) DNA-binding proteins (SSBs) stabilize ssDNA intermediates formed during genome maintenance reactions in Bacteria. SSBs also recruit proteins important for these processes through direct SSB-protein interactions, including proteins involved in DNA replication restart and recombination processes. SSBs are composed of an N-terminal oligomerization and ssDNA-binding domain, a C-terminal acidic tip that mediates SSB-protein interactions, and an internal intrinsically disordered linker (IDL). Deletions and insertions into the IDL are well tolerated with few phenotypes, although the largest deletions and insertions exhibit some sensitivity to DNA-damaging agents. To define specific DNA metabolism processes dependent on IDL length, ssb mutants that lack 16, 26, 37, or 47 residues of the 57-residue IDL were tested for synthetic phenotypes with mutations in DNA replication restart or recombination genes. We also tested the impact of integrating a fluorescent domain within the SSB IDL using an ssb::mTur2 insertion mutation. Only the largest deletion tested or the insertion mutation causes sensitivity in any of the pathways. Mutations in two replication restart pathways (PriA-B1 and PriA-C) showed synthetic lethalities or small colony phenotypes with the largest deletion or insertion mutations. Recombination gene mutations del(recBCD) and del(ruvABC) show synthetic phenotypes only when combined with the largest ssb deletion. These results suggest that a minimum IDL length is important in some genome maintenance reactions in Escherichia coli. These include pathways involving PriA-PriB1, PriA-PriC, RecFOR, and RecG. The mTur2 insertion in the IDL may also affect SSB interactions in some processes, particularly the PriA-PriB1 and PriA-PriC replication restart pathways.IMPORTANCEssb is essential in Escherichia coli due to its roles in protecting ssDNA and coordinating genome maintenance events. While the DNA-binding core and acidic tip have well-characterized functions, the purpose of the intrinsically disordered linker (IDL) is poorly understood. In vitro studies have revealed that the IDL is important for cooperative ssDNA binding and phase separation. However, single-stranded (ss) DNA-binding protein (SSB) variants with large deletions and insertions in the IDL support normal cell growth. We find that the PriA-PriB1 and PriA-C replication restart, as well as the RecFOR- and RecG-dependent recombination, pathways are sensitive to IDL length. This suggests that cooperativity, phase separation, or a longer spacer between the core and acidic tip of SSB may be important for specific cellular functions.

Oncogenic ETS fusions promote DNA damage and proinflammatory responses via pericentromeric RNAs in extracellular vesicles.

Aberrant expression of the E26 transformation-specific (ETS) transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS:FLI1 and EWS:ERG fusions in Ewing sarcoma (EwS) and TMPRSS2:ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS:FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV, and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs), and are highly elevated in plasma of patients with EwS with metastatic disease. High human satellite 2 and 3 (HSAT2,3) levels in EWS:FLI1- or ERG-expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle, and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA, and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs that transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

Genomic attributes of airway commensal bacteria and mucosa.

Microbial communities at the airway mucosal barrier are conserved and highly ordered, in likelihood reflecting co-evolution with human host factors. Freed of selection to digest nutrients, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the initial results of systematic culture and whole-genome sequencing of the thoracic airway bacteria, identifying 52 novel species amongst 126 organisms that constitute 75% of commensals typically present in heathy individuals. Clinically relevant genes encode antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. Using whole-genome content we identify dysbiotic features that may influence asthma and chronic obstructive pulmonary disease. We match isolate gene content to transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways to sustain host interactions with microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosa in lung diseases of global significance.

Controlling Genome Topology with Sequences that Trigger Post-replication Gap Formation During Replisome Passage: The E. coli RRS Elements.

We report that the Escherichia coli chromosome includes novel GC-rich genomic structural elements that trigger formation of post-replication gaps upon replisome passage. The two nearly perfect 222 bp repeats, designated Replication Risk Sequences or RRS, are each 650 kb from the terminus sequence dif and flank the Ter macrodomain. RRS sequence and positioning is highly conserved in enterobacteria. At least one RRS appears to be essential unless a 200 kb region encompassing one of them is amplified. The RRS contain a G-quadruplex on the lagging strand which impedes DNA polymerase extension producing lagging strand ssDNA gaps, ≤2000 bp long, upon replisome passage. Deletion of both RRS elements has substantial effects on global genome structure and topology. We hypothesize that RRS elements serve as topological relief valves during chromosome replication and segregation. There have been no screens for genomic sequences that trigger transient gap formation. Functional analogs of RRS could be widespread, possibly including some enigmatic G-quadruplexes in eukaryotes.

Identification of recG genetic interactions in Escherichia coli by transposon sequencing.

IMPORTANCE: DNA damage and subsequent DNA repair processes are mutagenic in nature and an important driver of evolution in prokaryotes, including antibiotic resistance development. Genetic screening approaches, such as transposon sequencing (Tn-seq), have provided important new insights into gene function and genetic relationships. Here, we employed Tn-seq to gain insight into the function of the recG gene, which renders Escherichia coli cells moderately sensitive to a variety of DNA-damaging agents when they are absent. The reported recG genetic interactions can be used in combination with future screens to aid in a more complete reconstruction of DNA repair pathways in bacteria.

Climate trends and maize production nexus in Mississippi: empirical evidence from ARDL modelling.

Climate change poses a significant threat to agriculture. However, climatic trends and their impact on Mississippi (MS) maize (Zea mays L.) are unknown. The objectives were to: (i) analyze trends in climatic variables (1970 to 2020) using Mann-Kendall and Sen slope method, (ii) quantify the impact of climate change on maize yield in short and long run using the auto-regressive distributive lag (ARDL) model, and (iii) categorize the critical months for maize-climate link using Pearson's correlation matrix. The climatic variables considered were maximum temperature (Tmax), minimum temperature (Tmin), diurnal temperature range (DTR), precipitation (PT), relative humidity (RH), and carbon emissions (CO2). The pre-analysis, post-analysis, and model robustness statistical tests were verified, and all conditions were met. A significant upward trend in Tmax (0.13 °C/decade), Tmin (0.27 °C/decade), and CO2 (5.1 units/decade), and a downward trend in DTR ( - 0.15 °C/decade) were noted. The PT and RH insignificantly increased by 4.32 mm and 0.11% per decade, respectively. The ARDL model explained 76.6% of the total variations in maize yield. Notably, the maize yield had a negative correlation with Tmax for June, and July, with PT in August, and with DTR for June, July, and August, whereas a positive correlation was noted with Tmin in June, July, and August. Overall, a unit change in Tmax reduced the maize yield by 7.39% and 26.33%, and a unit change in PT reduced it by 0.65% and 2.69% in the short and long run, respectively. However, a unit change in Tmin, and CO2 emissions increased maize yield by 20.68% and 0.63% in the long run with no short run effect. Overall, it is imperative to reassess the agronomic management strategies, developing and testing cultivars adaptable to the revealed climatic trend, with ability to withstand severe weather conditions in ensuring sustainable maize production.

Climate trends and soybean production since 1970 in Mississippi: Empirical evidence from ARDL model.

Studying historical response of crops to weather conditions at a finer scale is essential for devising agricultural strategies tailored to expected climate changes. However, determining the relationship between crop and climate in Mississippi (MS) remains elusive. Therefore, this research attempted to i) estimate climate trends between 1970 and 2020 in MS during the soybean growing season (SGS) using the Mann-Kendall and Sen slope method, ii) calculate the impact of climate change on soybean yield using an auto-regressive distributive lag (ARDL) econometric model, and iii) identify the most critical months from a crop-climate perspective by generating a correlation between the detrended yield and the monthly average for each climatic variable. Specific variables considered were maximum temperature (Tmax), minimum temperature (Tmin), diurnal temperature range (DTR), precipitation (PT), carbon dioxide emissions (CO2), and relative humidity (RH). All required diagnostic-tests i.e., pre-analysis, post-analysis, model-sensitivity, and assessing the models' goodness-of-fit were performed and statistical standards were met. A positive trend in Tmin (+0.25 °C/decade), and a negative trend in DTR (-0.18 °C/decade) was found. Although Tmax, PT, and RH showed non-significant trends, numerical changes were noted as +0.11 °C/decade, +3.03 mm/decade, and -0.06 %/decade, respectively. Furthermore, soybean yield was positively correlated with Tmin (in June and September), PT (in July and August), and RH (in July), but negatively correlated with Tmax (in July and August) and DTR (in June, July, and August). Soybean yield was observed to be significantly reduced by 18.11 % over the long-term and by 5.51 % over the short-term for every 1 °C increase in Tmax. With every unit increase in Tmin and CO2 emissions, the yield of soybeans increased significantly by 7.76 % and 3.04 %, respectively. Altogether, soybeans in MS exhibited variable sensitivity to short- and long-terms climatic changes. The results highlight the importance of testing climate-resilient agronomic practices and cultivars that encompass asymmetric sensitivities in response to climatic conditions of MS.

Delayed acquisition of airway commensals in antibiotic naïve children and its relationship with wheezing in rural Ecuador.

Introduction: The hygiene hypothesis identified a relationship between living in rural areas and acquiring protective environmental factors against the development of asthma and atopy. In our previous study, we found a correlation between particular bacterial species and early-onset wheezing in infants from the rural tropics of Ecuador who were corticosteroid-naïve and had limited antibiotic exposure. We now describe a longitudinal study of infants conducted to determine the age-related changes of the microbiome and its relationship with wheezing. Methods: We performed an amplicon sequencing of the 16S rRNA bacterial gene from the oropharyngeal samples obtained from 110 infants who had a history of recurrent episodic wheezing sampled at different ages (7, 12, and 24 months) and compared it to the sequencing of the oropharyngeal samples from 150 healthy infants sampled at the same time points. Bioinformatic analyses were conducted using QIIME and R. Results: As expected, the microbiota diversity consistently increased as the infants grew older. Considering age-based microbiota changes, we found that infants with wheeze had significantly lower species richness than the healthy infants at 7 months, but not at 12 or 24 months. Most of the core and accessory organisms increased in abundance and prevalence with age, except for a few which decreased. At 7 months of age, infants with wheeze had notably higher levels of a single Streptococcus operational taxonomic unit and core microbiota member than controls. Conclusions: In a cohort with limited antibiotic and corticosteroid use, a progressively more complex and diverse respiratory microbial community develops with age. The respiratory microbiota in early life is altered in infants with wheeze, but this does not hold true in older infants.

Investigating Metabolic and Molecular Ecological Evolution of Opportunistic Pulmonary Fungal Coinfections: Protocol for a Laboratory-Based Cross-Sectional Study.

BACKGROUND: Fungal-bacterial cocolonization and coinfections pose an emerging challenge among patients suspected of having pulmonary tuberculosis (PTB); however, the underlying pathogenic mechanisms and microbiome interactions are poorly understood. Understanding how environmental microbes, such as fungi and bacteria, coevolve and develop traits to evade host immune responses and resist treatment is critical to controlling opportunistic pulmonary fungal coinfections. In this project, we propose to study the coexistence of fungal and bacterial microbial communities during chronic pulmonary diseases, with a keen interest in underpinning fungal etiological evolution and the predominating interactions that may exist between fungi and bacteria. OBJECTIVE: This is a protocol for a study aimed at investigating the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections through determining and characterizing the burden, etiological profiles, microbial communities, and interactions established between fungi and bacteria as implicated among patients with presumptive PTB. METHODS: This will be a laboratory-based cross-sectional study, with a sample size of 406 participants. From each participant, 2 sputa samples (one on-spot and one early morning) will be collected. These samples will then be analyzed for both fungal and bacterial etiology using conventional metabolic and molecular (intergenic transcribed spacer and 16S ribosomal DNA-based polymerase chain reaction) approaches. We will also attempt to design a genome-scale metabolic model for pulmonary microbial communities to analyze the composition of the entire microbiome (ie, fungi and bacteria) and investigate host-microbial interactions under different patient conditions. This analysis will be based on the interplays of genes (identified by metagenomics) and inferred from amplicon data and metabolites (identified by metabolomics) by analyzing the full data set and using specific computational tools. We will also collect baseline data, including demographic and clinical history, using a patient-reported questionnaire. Altogether, this approach will contribute to a diagnostic-based observational study. The primary outcome will be the overall fungal and bacterial diagnostic profile of the study participants. Other diagnostic factors associated with the etiological profile, such as incidence and prevalence, will also be analyzed using univariate and multivariate schemes. Odds ratios with 95% CIs will be presented with a statistical significance set at P<.05. RESULTS: The study has been approved by the Mbarara University Research Ethic Committee (MUREC1/7-07/09/20) and the Uganda National Council of Science and Technology (HS1233ES). Following careful scrutiny, the protocol was designed to enable patient enrollment, which began in March 2022 at Mbarara University Teaching Hospital. Data collection is ongoing and is expected to be completed by August 2023, and manuscripts will be submitted for publication thereafter. CONCLUSIONS: Through this protocol, we will explore the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections among patients with presumptive PTB. Establishing key fungal-bacterial cross-kingdom synergistic relationships is crucial for instituting fungal bacterial coinfecting etiology. TRIAL REGISTRATION: ISRCTN Registry ISRCTN33572982; https://tinyurl.com/caa2nw69. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/48014.

Combination Effects of Integrin-linked Kinase and Abelson Kinase Inhibition on Aberrant Mitosis and Cell Death in Glioblastoma Cells.

In cancer cells, inhibition of integrin-linked kinase (ILK) increases centrosome declustering causing mitotic arrest and cell death. Yet, not all cancer cells are susceptible to anti-ILK treatment alone. We investigate a combination drug strategy targeting ILK and another oncogenic kinase, Abelson kinase (ABL). Drug-concentration viability assays (i.e., MTT assays) indicate that ILK and ABL inhibitors in combination decreased the viability of glioblastoma cells over the ILK drug QLT-0267 alone. Combination strategies also increased aberrant mitoses and cell death over QLT-0267 alone. This was evident from an increase in mitotic arrest, apoptosis and a sub-G1 peak following FAC analysis. In vitro, ILK and ABL localized to the centrosome and the putative ILK kinase domain was important for this localization. Increased levels of cytosolic ABL are associated with its transformative abilities. ILK inhibitor effects on survival correlated with its ability to decrease cytosolic ABL levels and inhibit ABL's localization to mitotic centrosomes in glioblastoma cells. ILK inhibitor effects on ABL's centrosomal localization were reversed by the proteasomal inhibitor MG132 (a drug that inhibits ABL degradation). These results indicate that ILK regulates ABL at mitotic centrosomes and that combination treatments targeting ILK and ABL are more effective then QLT-0267 alone at decreasing the survival of dividing glioblastoma cells.

Interaction with single-stranded DNA-binding protein modulates Escherichia coli RadD DNA repair activities.

The bacterial RadD enzyme is important for multiple genome maintenance pathways, including RecA DNA strand exchange and RecA-independent suppression of DNA crossover template switching. However, much remains unknown about the precise roles of RadD. One potential clue into RadD mechanisms is its direct interaction with the single-stranded DNA binding protein (SSB), which coats single-stranded DNA exposed during genome maintenance reactions in cells. Interaction with SSB stimulates the ATPase activity of RadD. To probe the mechanism and importance of RadD-SSB complex formation, we identified a pocket on RadD that is essential for binding SSB. In a mechanism shared with many other SSB-interacting proteins, RadD uses a hydrophobic pocket framed by basic residues to bind the C-terminal end of SSB. We found that RadD variants that substitute acidic residues for basic residues in the SSB binding site impair RadD:SSB complex formation and eliminate SSB stimulation of RadD ATPase activity in vitro. Additionally, mutant Escherichia coli strains carrying charge reversal radD changes display increased sensitivity to DNA damaging agents synergistically with deletions of radA and recG, although the phenotypes of the SSB-binding radD mutants are not as severe as a full radD deletion. This suggests that cellular RadD requires an intact interaction with SSB for full RadD function.

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.

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.

Generation and Repair of Postreplication Gaps in Escherichia coli.

When replication forks encounter template lesions, one result is lesion skipping, where the stalled DNA polymerase transiently stalls, disengages, and then reinitiates downstream to leave the lesion behind in a postreplication gap. Despite considerable attention in the 6 decades since postreplication gaps were discovered, the mechanisms by which postreplication gaps are generated and repaired remain highly enigmatic. This review focuses on postreplication gap generation and repair in the bacterium Escherichia coli. New information to address the frequency and mechanism of gap generation and new mechanisms for their resolution are described. There are a few instances where the formation of postreplication gaps appears to be programmed into particular genomic locations, where they are triggered by novel genomic elements.

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.

RecA and SSB genome-wide distribution in ssDNA gaps and ends in Escherichia coli.

Single-stranded DNA (ssDNA) gapped regions are common intermediates in DNA transactions. Using a new non-denaturing bisulfite treatment combined with ChIP-seq, abbreviated 'ssGap-seq', we explore RecA and SSB binding to ssDNA on a genomic scale in E. coli in a wide range of genetic backgrounds. Some results are expected. During log phase growth, RecA and SSB assembly profiles coincide globally, concentrated on the lagging strand and enhanced after UV irradiation. Unexpected results also abound. Near the terminus, RecA binding is favored over SSB, binding patterns change in the absence of RecG, and the absence of XerD results in massive RecA assembly. RecA may substitute for the absence of XerCD to resolve chromosome dimers. A RecA loading pathway may exist that is independent of RecBCD and RecFOR. Two prominent and focused peaks of RecA binding revealed a pair of 222 bp and GC-rich repeats, equidistant from dif and flanking the Ter domain. The repeats, here named RRS for replication risk sequence, trigger a genomically programmed generation of post-replication gaps that may play a special role in relieving topological stress during replication termination and chromosome segregation. As demonstrated here, ssGap-seq provides a new window on previously inaccessible aspects of ssDNA metabolism.

The "Ins and Outs" of Prostate Specific Membrane Antigen (PSMA) as Specific Target in Prostate Cancer Therapy.

Prostate-specific membrane antigen (PSMA) is expressed in epithelial cells of the prostate gland and is strongly upregulated in prostatic adenocarcinoma, with elevated expression correlating with metastasis, progression, and androgen independence. Because of its specificity, PSMA is a major target of prostate cancer therapy; however, detectable levels of PSMA are also found in other tissues, especially in salivary glands and kidney, generating bystander damage of these tissues. Antibody target therapy has been used with relative success in reducing tumor growth and prostate specific antigen (PSA) levels. However, since antibodies are highly stable in plasma, they have prolonged time in circulation and accumulate in organs with an affinity for antibodies such as bone marrow. For that reason, a second generation of PSMA targeted therapeutic agents has been developed. Small molecules and minibodies have had promising clinical trial results, but concerns about their specificity had arisen with side effects due to accumulation in salivary glands and kidneys. Herein we study the specificity of small molecules and minibodies that are currently being clinically tested. We observed a high affinity of these molecules for PSMA in prostate, kidney and salivary gland, suggesting that their effect is not prostate specific. The search for specific prostate target agents must continue so as to optimally treat patients with prostate cancer, while minimizing deleterious effects in other PSMA expressing tissues.