RecA-dependent or independent recombination of plasmid DNA generates a conflict with the host EcoKI immunity by launching restriction alleviation.

Bacterial defence systems are tightly regulated to avoid autoimmunity. In Type I restriction-modification (R-M) systems, a specific mechanism called restriction alleviation (RA) controls the activity of the restriction module. In the case of the Escherichia coli Type I R-M system EcoKI, RA proceeds through ClpXP-mediated proteolysis of restriction complexes bound to non-methylated sites that appear after replication or reparation of host DNA. Here, we show that RA is also induced in the presence of plasmids carrying EcoKI recognition sites, a phenomenon we refer to as plasmid-induced RA. Further, we show that the anti-restriction behavior of plasmid-borne non-conjugative transposons such as Tn5053, previously attributed to their ardD loci, is due to plasmid-induced RA. Plasmids carrying both EcoKI and Chi sites induce RA in RecA- and RecBCD-dependent manner. However, inactivation of both RecA and RecBCD restores RA, indicating that there exists an alternative, RecA-independent, homologous recombination pathway that is blocked in the presence of RecBCD. Indeed, plasmid-induced RA in a RecBCD-deficient background does not depend on the presence of Chi sites. We propose that processing of random dsDNA breaks in plasmid DNA via homologous recombination generates non-methylated EcoKI sites, which attract EcoKI restriction complexes channeling them for ClpXP-mediated proteolysis.

E. coli RecB Nuclease Domain Regulates RecBCD Helicase Activity but not Single Stranded DNA Translocase Activity.

Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as occurring by the ATPase motors mechanically pulling the DNA duplex across a wedge domain in the helicase, biochemical data show that processive DNA unwinding by E. coli RecBCD helicase can occur in the absence of ssDNA translocation by the canonical RecB and RecD motors. Here we show that DNA unwinding is not a simple consequence of ssDNA translocation by the motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecBΔNucCD) unwinds dsDNA at significantly slower rates than RecBCD, while the ssDNA translocation rate is unaffected. This effect is primarily due to the absence of the nuclease domain since a nuclease-dead mutant (RecBD1080ACD), which retains the nuclease domain, showed no change in ssDNA translocation or dsDNA unwinding rates relative to RecBCD on short DNA substrates (≤60 base pairs). Hence, ssDNA translocation is not rate-limiting for DNA unwinding. RecBΔNucCD also initiates unwinding much slower than RecBCD from a blunt-ended DNA. RecBΔNucCD also unwinds DNA ∼two-fold slower than RecBCD on long DNA (∼20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecBD1080ACD unwinding are intermediate between RecBCD and RecBΔNucCD. Surprisingly, significant pauses in DNA unwinding occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, possibly allosterically and that RecBΔNucCD may mimic a post-chi state of RecBCD.

RecBCD enzyme: mechanistic insights from mutants of a complex helicase-nuclease.

SUMMARYRecBCD enzyme is a multi-functional protein that initiates the major pathway of homologous genetic recombination and DNA double-strand break repair in Escherichia coli. It is also required for high cell viability and aids proper DNA replication. This 330-kDa, three-subunit enzyme is one of the fastest, most processive helicases known and contains a potent nuclease controlled by Chi sites, hotspots of recombination, in DNA. RecBCD undergoes major changes in activity and conformation when, during DNA unwinding, it encounters Chi (5'-GCTGGTGG-3') and nicks DNA nearby. Here, we discuss the multitude of mutations in each subunit that affect one or another activity of RecBCD and its control by Chi. These mutants have given deep insights into how the multiple activities of this complex enzyme are coordinated and how it acts in living cells. Similar studies could help reveal how other complex enzymes are controlled by inter-subunit interactions and conformational changes.

chi sequences switch the RecBCD helicase-nuclease complex from degradative to replicative modes during the completion of DNA replication.

Accurately completing DNA replication when two forks converge is essential to genomic stability. The RecBCD helicase-nuclease complex plays a central role in completion by promoting resection and joining of the excess DNA created when replisomes converge. chi sequences alter RecBCD activity and localize with crossover hotspots during sexual events in bacteria, yet their functional role during chromosome replication remains unknown. Here, we use two-dimensional agarose gel analysis to show that chi induces replication on substrates containing convergent forks. The induced replication is processive but uncoupled with respect to leading and lagging strand synthesis and can be suppressed by ter sites which limit replisome progression. Our observations demonstrate that convergent replisomes create a substrate that is processed by RecBCD and that chi, when encountered, switches RecBCD from a degradative to replicative function. We propose that chi serves to functionally differentiate DNA ends created during completion, which require degradation, from those created by chromosomal double-strand breaks, which require resynthesis.

A flexible RecC surface loop required for Chi hotspot control of RecBCD enzyme.

Escherichia coli RecBCD helicase-nuclease promotes vital homologous recombination-based repair of DNA double-strand breaks. The RecB nuclease domain (Nuc) is connected to the RecB helicase domain by a 19-amino-acid tether. When DNA binds to RecBCD, published evidence suggests that Nuc moves ∼50 Šfrom the exit of a RecC tunnel, from which the 3'-ended strand emerges during unwinding, to a distant position on RecC's surface. During subsequent ATP-dependent unwinding of DNA, Nuc nicks the 3'-ended strand near 5'-GCTGGTGG-3' (Chi recombination hotspot). Here, we test our model of Nuc swinging on the tether from the RecC tunnel exit to the RecC distant surface and back to the RecC tunnel exit to cut at Chi. We identify positions in a flexible surface loop on RecC and on RecB Nuc with complementary charges, mutation of which strongly reduces but does not eliminate Chi hotspot activity in cells. The recC loop mutation interacts with recB mutations hypothesized to be in the Chi-activated intramolecular signal transduction pathway; the double mutants, but not the single mutants, eliminate Chi hotspot activity. A RecC amino acid near the flexible loop is also essential for full Chi activity; its alteration likewise synergizes with a signal transduction mutation to eliminate Chi activity. We infer that altering the RecC surface loop reduces coordination among the subunits, which is critical for Chi hotspot activity. We discuss other RecBCD mutants with related properties.

Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub.

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.

RecBCD enzyme and Chi recombination hotspots as determinants of self vs. non-self: Myths and mechanisms.

Bacteria face a challenge when DNA enters their cells by transformation, mating, or phage infection. Should they treat this DNA as an invasive foreigner and destroy it, or consider it one of their own and potentially benefit from incorporating new genes or alleles to gain useful functions? It is frequently stated that the short nucleotide sequence Chi (5' GCTGGTGG 3'), a hotspot of homologous genetic recombination recognized by Escherichia coli's RecBCD helicase-nuclease, allows E. coli to distinguish its DNA (self) from any other DNA (non-self) and to destroy non-self DNA, and that Chi is "over-represented" in the E. coli genome. We show here that these latter statements (dogmas) are not supported by available evidence. We note Chi's wide-spread occurrence and activity in distantly related bacterial species and phages. We illustrate multiple, highly non-random features of the genomes of E. coli and coliphage P1 that account for Chi's high frequency and genomic position, leading us to propose that P1 selects for Chi's enhancement of recombination, whereas E. coli selects for the preferred codons in Chi. We discuss other, previously described mechanisms for self vs. non-self determination involving RecBCD and for RecBCD's destruction of DNA that cannot recombine, whether foreign or domestic, with or without Chi.

RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity.

The RecA protein and RecBCD complex are key bacterial components for the maintenance and repair of DNA. RecBCD is a helicase-nuclease that uses homologous recombination to resolve double-stranded DNA breaks. It also facilitates coating of single-stranded DNA with RecA to form RecA filaments, a vital step in the double-stranded break DNA repair pathway. However, questions remain about the mechanistic roles of RecA and RecBCD in live cells. Here, we use millisecond super-resolved fluorescence microscopy to pinpoint the spatial localization of fluorescent reporters of RecA or RecB at physiological levels of expression in individual live Escherichia coli cells. By introducing the DNA cross-linker mitomycin C, we induce DNA damage and quantify the resulting steady state changes in stoichiometry, cellular protein copy number and molecular mobilities of RecA and RecB. We find that both proteins accumulate in molecular hotspots to effect repair, resulting in RecA stoichiometries equivalent to several hundred molecules that assemble largely in dimeric subunits before DNA damage, but form periodic subunits of approximately 3-4 molecules within mature filaments of several thousand molecules. Unexpectedly, we find that the physiologically predominant forms of RecB are not only rapidly diffusing monomers, but slowly diffusing dimers.

Auxiliary ATP binding sites support DNA unwinding by RecBCD.

The RecBCD helicase initiates double-stranded break repair in bacteria by processively unwinding DNA with a rate approaching ∼1,600 bp·s-1, but the mechanism enabling such a fast rate is unknown. Employing a wide range of methodologies - including equilibrium and time-resolved binding experiments, ensemble and single-molecule unwinding assays, and crosslinking followed by mass spectrometry - we reveal the existence of auxiliary binding sites in the RecC subunit, where ATP binds with lower affinity and distinct chemical interactions as compared to the known catalytic sites. The essentiality and functionality of these sites are demonstrated by their impact on the survival of E.coli after exposure to damage-inducing radiation. We propose a model by which RecBCD achieves its optimized unwinding rate, even when ATP is scarce, by using the auxiliary binding sites to increase the flux of ATP to its catalytic sites.

Viral recombination systems limit CRISPR-Cas targeting through the generation of escape mutations.

CRISPR-Cas systems provide immunity to bacteria by programing Cas nucleases with RNA guides that recognize and cleave infecting viral genomes. Bacteria and their viruses each encode recombination systems that could repair the cleaved viral DNA. However, it is unknown whether and how these systems can affect CRISPR immunity. Bacteriophage λ uses the Red system (gam-exo-bet) to promote recombination between related phages. Here, we show that λ Red also mediates evasion of CRISPR-Cas targeting. Gam inhibits the host E. coli RecBCD recombination system, allowing recombination and repair of the cleaved DNA by phage Exo-Beta, which promotes the generation of mutations within the CRISPR target sequence. Red recombination is strikingly more efficient than the host's RecBCD-RecA in the production of large numbers of phages that escape CRISPR targeting. These results reveal a role for Red-like systems in the protection of bacteriophages against sequence-specific nucleases, which may facilitate their spread across viral genomes.

Heterogeneity in E. coli RecBCD Helicase-DNA Binding and Base Pair Melting.

E. coli RecBCD, a helicase/nuclease involved in double stranded (ds) DNA break repair, binds to a dsDNA end and melts out several DNA base pairs (bp) using only its binding free energy. We examined RecBCD-DNA initiation complexes using thermodynamic and structural approaches. Measurements of enthalpy changes for RecBCD binding to DNA ends possessing pre-melted ssDNA tails of increasing length suggest that RecBCD interacts with ssDNA as long as 17-18 nucleotides and can melt at least 10-11 bp upon binding a blunt DNA end. Cryo-EM structures of RecBCD alone and in complex with a blunt-ended dsDNA show significant conformational heterogeneities associated with the RecB nuclease domain (RecBNuc) and the RecD subunit. In the absence of DNA, 56% of RecBCD molecules show no density for the RecB nuclease domain, RecBNuc, and all RecBCD molecules show only partial density for RecD. DNA binding reduces these conformational heterogeneities, with 63% of the molecules showing density for both RecD and RecBNuc. This suggests that the RecBNuc domain is dynamic and influenced by DNA binding. The major RecBCD-DNA structural class in which RecBNuc is docked onto RecC shows melting of at least 11 bp from a blunt DNA end, much larger than previously observed. A second structural class in which RecBNuc is not docked shows only four bp melted suggesting that RecBCD complexes transition between states with different extents of DNA melting and that the extent of melting regulates initiation of helicase activity.

Targeting the bacterial SOS response for new antimicrobial agents: drug targets, molecular mechanisms and inhibitors.

Antimicrobial resistance is a pressing threat to global health, with multidrug-resistant pathogens becoming increasingly prevalent. The bacterial SOS pathway functions in response to DNA damage that occurs during infection, initiating several pro-survival and resistance mechanisms, such as DNA repair and hypermutation. This makes SOS pathway components potential targets that may combat drug-resistant pathogens and decrease resistance emergence. This review discusses the mechanism of the SOS pathway; the structure and function of potential targets AddAB, RecBCD, RecA and LexA; and efforts to develop selective small-molecule inhibitors of these proteins. These inhibitors may serve as valuable tools for target validation and provide the foundations for desperately needed novel antibacterial therapeutics.

Exonuclease 5 is dispensable for meiotic progression and male fertility in mouse.

Exonuclease 5 (Exo5) belongs to a class of bi-directional, ssDNA-specific exonucleases that mainly involved in the DNA repair pathways. Exo5 has been reported to be crucial for DNA- DNA mismatch repair (MMR) in several human cell lines. However, its in vivo function in mammals still needs to be explored. Thus, to study the in vivo role of Exo5 in spermatogenesis, Exo5 knockout mice were generated using CRISPR/Cas9 technology. Unexpectedly, we found that the knockout mice are fertile despite a slight decrease in sperm count. Furthermore, Exo5-/- mice showed no detectable developmental anomalies, exhibited no remarkable differences in the epididymal histology and testis/body weight ratio. Moreover, cytological investigations on meiocytes revealed non-significant differences in chromosomal synapsis, recombination, and meiotic progression of prophase I, further demonstrating that Exo5 has no essential role in spermatogenesis in mice under normal breeding conditions. Collectively, these data indicate that Exo5 is dispensable for meiotic progression and fertility in mice.

Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling.

Repair of broken DNA by homologous recombination requires coordinated enzymatic reactions to prepare it for interaction with intact DNA. The multiple activities of enterobacterial RecBCD helicase-nuclease are coordinated by Chi recombination hotspots (5' GCTGGTGG 3') recognized during DNA unwinding. Chi is recognized in a tunnel in RecC but activates the RecB nuclease, > 25 Ǻ away. How the Chi-dependent signal travels this long distance has been unknown. We found a Chi hotspot-deficient mutant in the RecB helicase domain located > 45 Ǻ from both the Chi-recognition site and the nuclease active site. This unexpected observation led us to find additional mutations that reduced or eliminated Chi hotspot activity in each subunit and widely scattered throughout RecBCD. Each mutation alters the intimate contact between one or another pair of subunits in crystal or cryoEM structures of RecBCD bound to DNA. Collectively, these mutations span a path about 185 Ǻ long from the Chi recognition site to the nuclease active site. We discuss these surprising results in the context of an intramolecular signal transduction accounting for many previous observations.

An Exonuclease V-qPCR Assay to Analyze the State of the Human Papillomavirus 16 Genome in Cell Lines and Tissues.

Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC. Basic Protocol: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cells Support Protocol: Exonuclease V analysis of HPV16 genome configuration in tissues Alternate Protocol: Determining HPV integration type or integrity of HPV episome.

Distribution of RecBCD and AddAB recombination-associated genes among bacteria in 33 phyla.

Homologous recombination plays key roles in fundamental processes such as recovery from DNA damage and in bacterial horizontal gene transfer, yet there are still open questions about the distribution of recognized components of recombination machinery among bacteria and archaea. RecBCD helicase-nuclease plays a central role in recombination among Gammaproteobacteria like Escherichia coli; while bacteria in other phyla, like the Firmicute Bacillus subtilis, use the related AddAB complex. The activity of at least some of these complexes is controlled by short DNA sequences called crossover hotspot instigator (Chi) sites. When RecBCD or AddAB complexes encounter an autologous Chi site during unwinding, they introduce a nick such that ssDNA with a free end is available to invade another duplex. If homologous DNA is present, RecA-dependent homologous recombination is promoted; if not (or if no autologous Chi site is present) the RecBCD/AddAB complex eventually degrades the DNA. We examined the distribution of recBCD and addAB genes among bacteria, and sought ways to distinguish them unambiguously. We examined bacterial species among 33 phyla, finding some unexpected distribution patterns. RecBCD and addAB are less conserved than recA, with the orthologous recB and addA genes more conserved than the recC or addB genes. We were able to classify RecB vs. AddA and RecC vs. AddB in some bacteria where this had not previously been done. We used logo analysis to identify sequence segments that are conserved, but differ between the RecBC and AddAB proteins, to help future differentiation between members of these two families.

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.

Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state.

Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5'-ended strand and its slower RecB helicase on the 3'-ended strand. At Chi hotspots (5' GCTGGTGG 3'), RecB's nuclease cuts the 3'-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB's ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.

Genome Maintenance Proteins Modulate Autoimmunity Mediated Primed Adaptation by the Escherichia coli Type I-E CRISPR-Cas System.

Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders' genomes into CRISPR arrays. The acquisition of fragments of host genomes leads to autoimmunity and may drive chromosomal rearrangements, negative cell selection, and influence bacterial evolution. In this study, we investigated the role of proteins involved in genome stability maintenance in spacer acquisition by the Escherichia coli type I-E CRISPR-Cas system targeting its own genome. We show here, that the deletion of recJ decreases adaptation efficiency and affects accuracy of spacers incorporation into CRISPR array. Primed adaptation efficiency is also dramatically inhibited in double mutants lacking recB and sbcD but not in single mutants suggesting independent involvement and redundancy of RecBCD and SbcCD pathways in spacer acquisition. While the presence of at least one of two complexes is crucial for efficient primed adaptation, RecBCD and SbcCD affect the pattern of acquired spacers. Overall, our data suggest distinct roles of the RecBCD and SbcCD complexes and of RecJ in spacer precursor selection and insertion into CRISPR array and highlight the functional interplay between CRISPR-Cas systems and host genome maintenance mechanisms.

RecBCD, SbcCD and ExoI process a substrate created by convergent replisomes to complete DNA replication.

The accurate completion of DNA replication on the chromosome requires RecBCD and structure specific SbcCD and ExoI nucleases. However, the substrates and mechanism by which this reaction occurs remains unknown. Here we show that these completion enzymes operate on plasmid substrates containing two replisomes, but are not required for plasmids containing one replisome. Completion on the two-replisome plasmids requires RecBCD, but does not require RecA and no broken intermediates accumulate in its absence, indicating that the completion reaction occurs normally in the absence of any double-strand breaks. Further, similar to the chromosome, we show that when the normal completion reaction is prevented, an aberrant RecA-mediated recombination process leads to amplifications that drive most of the instabilities associated with the two-replisome substrates. The observations imply that the substrate SbcCD, ExoI and RecBCD act upon in vivo is created specifically by two convergent replisomes, and demonstrate that the function of RecBCD in completing replication is independent of double-strand break repair, and likely promotes joining of the strands of the convergent replication forks.