Formation of memory assemblies through the DNA-sensing TLR9 pathway.

As hippocampal neurons respond to diverse types of information1, a subset assembles into microcircuits representing a memory2. Those neurons typically undergo energy-intensive molecular adaptations, occasionally resulting in transient DNA damage3-5. Here we found discrete clusters of excitatory hippocampal CA1 neurons with persistent double-stranded DNA (dsDNA) breaks, nuclear envelope ruptures and perinuclear release of histone and dsDNA fragments hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of TLR9 signalling and accumulation of centrosomal DNA damage repair complexes6. Neuron-specific knockdown of Tlr9 impaired memory while blunting contextual fear conditioning-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Notably, TLR9 had an essential role in centrosome function, including DNA damage repair, ciliogenesis and build-up of perineuronal nets. We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and TLR9-mediated repair, resulting in their recruitment to memory circuits. With compromised TLR9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments implicated in accelerated senescence, psychiatric disorders and neurodegenerative disorders. Maintaining the integrity of TLR9 inflammatory signalling thus emerges as a promising preventive strategy for neurocognitive deficits.

Sizing up to divide: mitotic cell-size control in fission yeast.

Schizosaccharomyces pombe is a good model to study cell-size control. These cells integrate size information into cell cycle controls at both the G1/S and G2/M transitions, although the primary control operates at the entry into mitosis. At G2/M there is both a size threshold, demonstrated by the fact that cells divide when they reach 14 µm in length, and also correction around this threshold, evident from the narrow distribution of sizes within a population. This latter property is referred to as size homeostasis. It has been argued that a population of cells accumulating mass in a linear fashion will have size homeostasis in the absence of size control, if cycle time is controlled by a fixed timer. Because fission yeast cells do not grow in a simple linear fashion, they require a size-sensing mechanism. However, current models do not fully describe all aspects of this control, especially the coordination of cell size with ploidy.

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.

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.

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.

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.

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.

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.

Functional quality of life among newly diagnosed young adult colorectal cancer survivors compared to older adults: results from the ColoCare Study.

PURPOSE: Colorectal cancer (CRC) incidence and mortality are increasing among young adults (YAs) aged 18-39. This study compared quality of life (QOL) between YA and older adult CRC survivors in the ColoCare Study. METHODS: Participants were grouped by age (years) as follows: 18-39 (YA), 40-49, 50-64, and 65 + . Functional QOL (physical, social, role, emotional, cognitive) and global QOL were assessed with the EORTC-QLQ-C30 at enrollment, 3, 6, and 12 months. Average scores were compared between groups over time using longitudinal mixed-effect modeling. Proportions with clinically meaningful QOL impairment were calculated using age-relevant thresholds and compared between groups over time using logistic regression with mixed effects. RESULTS: Participants (N = 1590) were n = 81 YAs, n = 196 aged 40-49, n = 627 aged 50-64, and n = 686 aged 65 + . Average physical function was better among YAs than participants aged 50-64 (p = 0.010) and 65 + (p < 0.001), and average social function was worse among YAs than aged 65 + (p = 0.046). Relative to YAs, all age groups were less likely to report clinically meaningful social dysfunction (aged 40-49 OR = 0.13, 95%CI = 0.06-0.29; aged 50-64 OR = 0.10, 95%CI = 0.05-0.21; aged 65 + OR = 0.07, 95%CI = 0.04-0.15) and role dysfunction (aged 40-49 OR = 0.36, 95%CI = 0.18-0.75; aged 50-64 OR = 0.41, 95%CI = 0.22-0.78; aged 65 + OR = 0.32, 95%CI = 0.17-0.61). Participants aged 40-49 were also less likely to report physical dysfunction (OR = 0.42, 95%CI = 0.19-0.93). CONCLUSION: YA CRC survivors reported better physical and worse social function compared to older CRC survivors, and YA CRC survivors were more likely to report clinically meaningful social, role, and physical disfunction. Future work should further investigate QOL using age-relevant benchmarks to inform best practices for CRC survivorship care. TRIAL REGISTRATION: NCT02328677, registered December 2014.

The ENGAGE study: a 3-arm randomized hybrid type 1 effectiveness and implementation study of an in-home, collaborative PCP model of remote telegenetic services to increase uptake of cancer genetic services in childhood cancer survivors.

BACKGROUND: Germline cancer genetic testing has become a standard evidence-based practice, with established risk reduction and screening guidelines for genetic carriers. Access to genetic services is limited in many places, which leaves many genetic carriers unidentified and at risk for late diagnosis of cancers and poor outcomes. This poses a problem for childhood cancer survivors, as this is a population with an increased risk for subsequent malignant neoplasms (SMN) due to cancer therapy or inherited cancer predisposition. The ENGaging and Activating cancer survivors in Genetic services (ENGAGE) study evaluates the effectiveness of an in-home, collaborative PCP model of remote telegenetic services to increase uptake of cancer genetic testing in childhood cancer survivors compared to usual care options for genetic testing. METHODS: The ENGAGE study is a 3-arm randomized hybrid type 1 effectiveness and implementation study within the Childhood Cancer Survivor Study population which tests a clinical intervention while gathering information on its delivery during the effectiveness trial and its potential for future implementation among 360 participants. Participants are randomized into three arms. Those randomized to Arm A receive genetic services via videoconferencing, those in Arm B receive these services by phone, and those randomized to Arm C will receive usual care services. DISCUSSION: With many barriers to accessing genetic services, innovative delivery models are needed to address this gap and increase uptake of genetic services. The ENGAGE study evaluates the effectiveness of an adapted model of remote delivery of genetic services to increase the uptake of recommended genetic testing in childhood cancer survivors. This study assesses the uptake in remote genetic services and identify barriers to uptake to inform future recommendations and a theoretically-informed process evaluation which can inform modifications to enhance dissemination beyond this study population and to realize the benefits of precision medicine. TRIAL REGISTRATION: This protocol was registered at clinicaltrials.gov (NCT04455698) on July 2, 2020.

The rarA gene as part of an expanded RecFOR recombination pathway: Negative epistasis and synthetic lethality with ruvB, recG, and recQ.

The RarA protein, homologous to human WRNIP1 and yeast MgsA, is a AAA+ ATPase and one of the most highly conserved DNA repair proteins. With an apparent role in the repair of stalled or collapsed replication forks, the molecular function of this protein family remains obscure. Here, we demonstrate that RarA acts in late stages of recombinational DNA repair of post-replication gaps. A deletion of most of the rarA gene, when paired with a deletion of ruvB or ruvC, produces a growth defect, a strong synergistic increase in sensitivity to DNA damaging agents, cell elongation, and an increase in SOS induction. Except for SOS induction, these effects are all suppressed by inactivating recF, recO, or recJ, indicating that RarA, along with RuvB, acts downstream of RecA. SOS induction increases dramatically in a rarA ruvB recF/O triple mutant, suggesting the generation of large amounts of unrepaired ssDNA. The rarA ruvB defects are not suppressed (and in fact slightly increased) by recB inactivation, suggesting RarA acts primarily downstream of RecA in post-replication gaps rather than in double strand break repair. Inactivating rarA, ruvB and recG together is synthetically lethal, an outcome again suppressed by inactivation of recF, recO, or recJ. A rarA ruvB recQ triple deletion mutant is also inviable. Together, the results suggest the existence of multiple pathways, perhaps overlapping, for the resolution or reversal of recombination intermediates created by RecA protein in post-replication gaps within the broader RecF pathway. One of these paths involves RarA.

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.

Associations of combined physical activity and body mass index groups with colorectal cancer survival outcomes.

BACKGROUND: Physical activity and BMI have been individually associated with cancer survivorship but have not yet been studied in combinations in colorectal cancer patients. Here, we investigate individual and combined associations of physical activity and BMI groups with colorectal cancer survival outcomes. METHODS: Self-reported physical activity levels (MET hrs/wk) were assessed using an adapted version of the International Physical Activity Questionnaire (IPAQ) at baseline in 931 patients with stage I-III colorectal cancer and classified into 'highly active' and'not-highly active'(≥ / < 18 MET hrs/wk). BMI (kg/m2) was categorized into 'normal weight', 'overweight', and 'obese'. Patients were further classified into combined physical activity and BMI groups. Cox-proportional hazard models with Firth correction were computed to assess associations [hazard ratio (HR), 95% profile HR likelihood confidence interval (95% CI) between individual and combined physical activity and BMI groups with overall and disease-free survival in colorectal cancer patients. RESULTS: 'Not-highly active' compared to 'highly active' and 'overweight'/ 'obese' compared to 'normal weight' patients had a 40-50% increased risk of death or recurrence (HR: 1.41 (95% CI: 0.99-2.06), p = 0.03; HR: 1.49 (95% CI: 1.02-2.21) and HR: 1.51 (95% CI: 1.02-2.26), p = 0.04, respectively). 'Not-highly active' patients had worse disease-free survival outcomes, regardless of their BMI, compared to 'highly active/normal weight' patients. 'Not-highly active/obese' patients had a 3.66 times increased risk of death or recurrence compared to 'highly active/normal weight' patients (HR: 4.66 (95% CI: 1.75-9.10), p = 0.002). Lower activity thresholds yielded smaller effect sizes. CONCLUSION: Physical activity and BMI were individually associated with disease-free survival among colorectal cancer patients. Physical activity seems to improve survival outcomes in patients regardless of their BMI.

RecA-independent recombination: Dependence on the Escherichia coli RarA protein.

Most, but not all, homologous genetic recombination in bacteria is mediated by the RecA recombinase. The mechanistic origin of RecA-independent recombination has remained enigmatic. Here, we demonstrate that the RarA protein makes a major enzymatic contribution to RecA-independent recombination. In particular, RarA makes substantial contributions to intermolecular recombination and to recombination events involving relatively short (<200 bp) homologous sequences, where RecA-mediated recombination is inefficient. The effects are seen here in plasmid-based recombination assays and in vivo cloning processes. Vestigial levels of recombination remain even when both RecA and RarA are absent. Additional pathways for RecA-independent recombination, possibly mediated by helicases, are suppressed by exonucleases ExoI and RecJ. Translesion DNA polymerases may also contribute. Our results provide additional substance to a previous report of a functional overlap between RecA and RarA.

Mismatches in resident and stranger serotonin transporter genotypes lead to escalated aggression, and the target for aggression is mediated by sex differences in male and female rhesus monkeys (Macaca mulatta).

A variety of studies show that the s-allele of the serotonin transporter genotype (5-HTT) is related to aggression. However, influences of sex and 5-HTT genotype of both subject and opponent have not received as much attention in aggression research. Using a nonhuman primate model, the present study explores differences in rates of aggression exhibited by 201 group-housed male and female rhesus monkeys (Macaca mulatta; 122 females; 79 males) exposed to an unfamiliar age- and sex-matched stranger while in the presence of other same-sex members of their social group. The study also assesses whether the rates of aggression increase when the home-cage resident, the unfamiliar stimulus animal, or both possess the short (s) allele of the 5-HTT. Results showed that, when compared to females, males exhibited higher rates of physical aggression toward the stranger, and when both the male resident and the male stranger possessed the s-allele, rates of physical aggression toward the stranger increased five-fold. Resident females also engaged in higher rates of physical aggression when they possessed the s-allele, although unlike the males, their physical aggression was directed toward familiar same-sex members of their social group. The findings of this study indicate that rates of physical aggression are modulated by 5-HTT resident and stranger suggest a role of sexual competition in the phenotype of the 5-HTT genotype. Importantly, when two males with impulse deficits, as a function of the s-allele, are placed together, rates of violence exhibited by the dyad escalate substantially.

Central nervous system monoamine metabolite response to alcohol exposure is associated with future alcohol intake in a nonhuman primate model (Macaca mulatta).

It is widely held that the central monoamine neurotransmitters modulate alcohol intake. Few studies, however, directly assess the relationship between baseline and alcohol-induced monoamine turnover, as well as the change from baseline, as predictors of alcohol intake. Using a nonhuman primate model, this study investigates baseline, alcohol-induced and alcohol-induced change in monoamine activity and their relationship with alcohol intake. Alcohol-naïve, adolescent rhesus macaques (Macaca mulatta, N = 114) were administered a standardized intravenous bolus of alcohol solution (16.8%, v/v) on two occasions, approximately 1 month apart. One month prior to and 1 h following each alcohol infusion, cisternal cerebrospinal fluid (CSF) was obtained and assayed for monoamine metabolite concentrations. Approximately 6-7 months later, subjects were allowed unfettered access to an aspartame-sweetened alcohol solution (8.4%, v/v) for 1 h/day, 5 days/week, over 5-7 weeks. Results showed strong positive correlations between baseline and post-infusion CSF monoamine metabolite concentrations, indicating a trait-like response. Low baseline and post-infusion serotonin and dopamine metabolite concentrations and a smaller change in serotonin and dopamine metabolites from one infusion to the next were associated with higher alcohol intake. Low baseline and post-infusion norepinephrine metabolite concentrations predicted high alcohol intake, but unlike the other monoamines, a greater change in norepinephrine metabolite concentrations from one infusion to the next was associated with higher alcohol intake. These findings suggest that individual differences in naturally occurring and alcohol-induced monoamine activity, as well as the change between exposures, are important modulators of initial alcohol consumption and may play a role in the risk for excessive alcohol intake.

Race and sex differences in HDL peroxide content among American adults with and without type 2 diabetes.

BACKGROUND: High-density lipoprotein (HDL) plays a critical role in protection against atherosclerosic and cardiovascular disease (ASCVD). In addition to contributing to clearing excess vascular cholesterol, HDL particles exhibit antioxidative functions, helping to attenuate adverse effects of oxidized low-density lipoproteins. However, these beneficial properties can be undermined by oxidative stress, inflammation, and unhealthy lifestyles and diet, as well as influenced by race and sex. Thus, when assessing cardiovascular risk, it is important to consider multifactorial aspects of HDL, including antioxidant activity rather than just total amount and type of HDL-cholesterol (HDL-C) particles. Because prior research showed HDL peroxide content (HDLperox) can be inversely associated with normal anti-oxidant HDL activity, elevated HDLperox may serve as a bioindicator of HDL dysfunction. METHODS: In this study, data from a large national cohort of Americans was utilized to determine the impact of sex, race, and diabetes status on HDLperox in middle-aged and older adults. A previously developed cell-free fluorometric method was utilized to quantify HDLperox in serum depleted of apo-B containing lipoproteins. RESULTS: In keeping with predictions, white men and diabetics exhibited HDLperox in the atypical upper range, suggestive of less functional HDL. White men had higher HDLperox levels than African American males (13.46 ± 6.10 vs. 10.88 ± 5.81, p < .001). There was also a significant main effect of type 2 diabetes (F(1,1901) = 14.9, p < .0001). Overall, African Americans evinced lower HDLperox levels, despite more obesity (10.3 ± 4.7 vs.11.81 ± 5.66 for Whites) suggesting that other aspects of lipid metabolism and psychosocial factors account for the higher prevalence of ASCVD in African Americans. CONCLUSION: This research helps to provide a more comprehensive understanding of HDL function in a racially and metabolically diverse adult population. HDLperox content was significantly different in adults with type 2 diabetes, and distinctive in nondiabetic White males, and suggests other processes account for the higher prevalence of ASCVD among African Americans.

Resolving Toxic DNA repair intermediates in every E. coli replication cycle: critical roles for RecG, Uup and RadD.

DNA lesions or other barriers frequently compromise replisome progress. The SF2 helicase RecG is a key enzyme in the processing of postreplication gaps or regressed forks in Escherichia coli. A deletion of the recG gene renders cells highly sensitive to a range of DNA damaging agents. Here, we demonstrate that RecG function is at least partially complemented by another SF2 helicase, RadD. A ΔrecGΔradD double mutant exhibits an almost complete growth defect, even in the absence of stress. Suppressors appear quickly, primarily mutations that compromise priA helicase function or recA promoter mutations that reduce recA expression. Deletions of uup (encoding the UvrA-like ABC system Uup), recO, or recF also suppress the ΔrecGΔradD growth phenotype. RadD and RecG appear to avoid toxic situations in DNA metabolism, either resolving or preventing the appearance of DNA repair intermediates produced by RecA or RecA-independent template switching at stalled forks or postreplication gaps. Barriers to replisome progress that require intervention by RadD or RecG occur in virtually every replication cycle. The results highlight the importance of the RadD protein for general chromosome maintenance and repair. They also implicate Uup as a new modulator of RecG function.

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.