Recent insights into eukaryotic double-strand DNA break repair unveiled by single-molecule methods.

Genome integrity and maintenance are essential for the viability of all organisms. A wide variety of DNA damage types have been described, but double-strand breaks (DSBs) stand out as one of the most toxic DNA lesions. Two major pathways account for the repair of DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Both pathways involve complex DNA transactions catalyzed by proteins that sequentially or cooperatively work to repair the damage. Single-molecule methods allow visualization of these complex transactions and characterization of the protein:DNA intermediates of DNA repair, ultimately allowing a comprehensive breakdown of the mechanisms underlying each pathway. We review current understanding of the HR and NHEJ responses to DSBs in eukaryotic cells, with a particular emphasis on recent advances through the use of single-molecule techniques.

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.

Long DNA constructs to study helicases and nucleic acid translocases using optical tweezers.

Single molecule biophysics experiments for the study of DNA-protein interactions usually require production of a homogeneous population of long DNA molecules with controlled sequence content and/or internal tertiary structures. Traditionally, Lambda phage DNA has been used for this purpose, but it is difficult to customize. In this article, we provide a detailed and simple protocol for cloning large (~25kbp) plasmids with bespoke sequence content, which can be used to generate custom DNA constructs for a range of single-molecule experiments. In particular, we focus on a procedure for making long single-stranded DNA (ssDNA) molecules, ssDNA-dsDNA hybrids and long DNA constructs with flaps, which are especially relevant for studying the activity of DNA helicases and translocases. Additionally, we describe how the modification of the free ends of such substrates can facilitate their binding to functionalized surfaces allowing immobilization and imaging using dual optical tweezers and confocal microscopy. Finally, we provide examples of how these DNA constructs have been applied to study the activity of human DNA helicase B (HELB). The techniques described herein are simple, versatile, adaptable, and accessible to any laboratory with access to standard molecular biology methods.

Human HELB is a processive motor protein that catalyzes RPA clearance from single-stranded DNA.

Human DNA helicase B (HELB) is a poorly characterized helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single-molecule approaches to characterize the biochemical activities of HELB protein with a particular focus on its interactions with Replication Protein A (RPA) and RPA­single-stranded DNA (ssDNA) filaments. HELB is a monomeric protein that binds tightly to ssDNA with a site size of ∼20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5' to 3' direction accompanied by the formation of DNA loops. HELB also displays classical helicase activity, but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA, which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from ssDNA. This activity, which can allow other proteins access to ssDNA intermediates despite their shielding by RPA, may underpin the diverse roles of HELB in cellular DNA transactions.

Targeted control of pneumolysin production by a mobile genetic element in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen that can cause severe invasive diseases such as pneumonia, septicaemia and meningitis. Young children are at a particularly high risk, with an estimated 3-4 million cases of severe disease and between 300 000 and 500 000 deaths attributable to pneumococcal disease each year. The haemolytic toxin pneumolysin (Ply) is a primary virulence factor for this bacterium, yet despite its key role in pathogenesis, immune evasion and transmission, the regulation of Ply production is not well defined. Using a genome-wide association approach, we identified a large number of potential affectors of Ply activity, including a gene acquired horizontally on the antibiotic resistance-conferring Integrative and Conjugative Element (ICE) ICESp23FST81. This gene encodes a novel modular protein, ZomB, which has an N-terminal UvrD-like helicase domain followed by two Cas4-like domains with potent ATP-dependent nuclease activity. We found the regulatory effect of ZomB to be specific for the ply operon, potentially mediated by its high affinity for the BOX repeats encoded therein. Using a murine model of pneumococcal colonization, we further demonstrate that a ZomB mutant strain colonizes both the upper respiratory tract and lungs at higher levels when compared to the wild-type strain. While the antibiotic resistance-conferring aspects of ICESp23FST81 are often credited with contributing to the success of the S. pneumoniae lineages that acquire it, its ability to control the expression of a major virulence factor implicated in bacterial transmission is also likely to have played an important role.

Analysis of the PcrA-RNA polymerase complex reveals a helicase interaction motif and a role for PcrA/UvrD helicase in the suppression of R-loops.

The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis for this interaction and its functional significance have remained unclear. In this work, we used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation complex in a manner dependent on protein:protein interaction with the conserved PcrA C-terminal Tudor domain. The helicase binds predominantly to two positions on the surface of RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert within the ß subunit which we identify as a helicase interaction motif present in many other PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop suppression factor that helps to minimize conflicts between transcription and other processes on DNA including replication.

A stapled peptide mimetic of the CtIP tetramerization motif interferes with double-strand break repair and replication fork protection.

Cancer cells display high levels of DNA damage and replication stress, vulnerabilities that could be exploited by drugs targeting DNA repair proteins. Human CtIP promotes homology-mediated repair of DNA double-strand breaks (DSBs) and protects stalled replication forks from nucleolytic degradation, thus representing an attractive candidate for targeted cancer therapy. Here, we establish a peptide mimetic of the CtIP tetramerization motif that inhibits CtIP activity. The hydrocarbon-stapled peptide encompassing amino acid residues 18 to 28 of CtIP (SP18-28) stably binds to CtIP tetramers in vitro and facilitates their aggregation into higher-order structures. Efficient intracellular uptake of SP18-28 abrogates CtIP localization to damaged chromatin, impairs DSB repair, and triggers extensive fork degradation. Moreover, prolonged SP18-28 treatment causes hypersensitivity to DNA-damaging agents and selectively reduces the viability of BRCA1-mutated cancer cell lines. Together, our data provide a basis for the future development of CtIP-targeting compounds with the potential to treat patients with cancer.

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover.

The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity-a prerequisite for understanding how biological systems work-has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.

Bulk and single-molecule analysis of a bacterial DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor.

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.

Direct removal of RNA polymerase barriers to replication by accessory replicative helicases.

Bacterial genome duplication and transcription require simultaneous access to the same DNA template. Conflicts between the replisome and transcription machinery can lead to interruption of DNA replication and loss of genome stability. Pausing, stalling and backtracking of transcribing RNA polymerases add to this problem and present barriers to replisomes. Accessory helicases promote fork movement through nucleoprotein barriers and exist in viruses, bacteria and eukaryotes. Here, we show that stalled Escherichia coli transcription elongation complexes block reconstituted replisomes. This physiologically relevant block can be alleviated by the accessory helicase Rep or UvrD, resulting in the formation of full-length replication products. Accessory helicase action during replication-transcription collisions therefore promotes continued replication without leaving gaps in the DNA. In contrast, DinG does not promote replisome movement through stalled transcription complexes in vitro. However, our data demonstrate that DinG operates indirectly in vivo to reduce conflicts between replication and transcription. These results suggest that Rep and UvrD helicases operate on DNA at the replication fork whereas DinG helicase acts via a different mechanism.

The molecular basis of protein toxin HicA-dependent binding of the protein antitoxin HicB to DNA.

Toxin-antitoxin (TA) systems are present in many bacteria and play important roles in bacterial growth, physiology, and pathogenicity. Those that are best studied are the type II TA systems, in which both toxins and antitoxins are proteins. The HicAB system is one of the prototypic TA systems, found in many bacterial species. Complex interactions between the protein toxin (HicA), the protein antitoxin (HicB), and the DNA upstream of the encoding genes regulate the activity of this system, but few structural details are available about how HicA destabilizes the HicB-DNA complex. Here, we determined the X-ray structures of HicB and the HicAB complex to 1.8 and 2.5 Å resolution, respectively, and characterized their DNA interactions. This revealed that HicB forms a tetramer and HicA and HicB form a heterooctameric complex that involves structural reorganization of the C-terminal (DNA-binding) region of HicB. Our observations indicated that HicA has a profound impact on binding of HicB to DNA sequences upstream of hicAB in a stoichiometric-dependent way. At low ratios of HicA:HicB, there was no effect on DNA binding, but at higher ratios, the affinity for DNA declined cooperatively, driving dissociation of the HicA:HicB:DNA complex. These results reveal the structural mechanisms by which HicA de-represses the HicB-DNA complex.

The 2B subdomain of Rep helicase links translocation along DNA with protein displacement.

Helicases catalyse DNA and RNA strand separation. Proteins bound to the nucleic acid must also be displaced in order to unwind DNA. This is exemplified by accessory helicases that clear protein barriers from DNA ahead of advancing replication forks. How helicases catalyse DNA unwinding is increasingly well understood but how protein displacement is achieved is unclear. Escherichia coli Rep accessory replicative helicase lacking one of its four subdomains, 2B, has been shown to be hyperactivated for DNA unwinding in vitro but we show here that RepΔ2B is, in contrast, deficient in displacing proteins from DNA. This defect correlates with an inability to promote replication of protein-bound DNA in vitro and lack of accessory helicase function in vivo. Defective protein displacement is manifested on double-stranded and single-stranded DNA. Thus binding and distortion of duplex DNA by the 2B subdomain ahead of the helicase is not the missing function responsible for this deficiency. These data demonstrate that protein displacement from DNA is not simply achieved by helicase translocation alone. They also imply that helicases may have evolved different specific features to optimise DNA unwinding and protein displacement, both of which are now recognised as key functions in all aspects of nucleic acid metabolism.

The structural basis for dynamic DNA binding and bridging interactions which condense the bacterial centromere.

The ParB protein forms DNA bridging interactions around parS to condense DNA and earmark the bacterial chromosome for segregation. The molecular mechanism underlying the formation of these ParB networks is unclear. We show here that while the central DNA binding domain is essential for anchoring at parS, this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensation in vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB-GFP foci formation in vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks in Bacillus subtilis.

The structure and function of an RNA polymerase interaction domain in the PcrA/UvrD helicase.

The PcrA/UvrD helicase functions in multiple pathways that promote bacterial genome stability including the suppression of conflicts between replication and transcription and facilitating the repair of transcribed DNA. The reported ability of PcrA/UvrD to bind and backtrack RNA polymerase (1,2) might be relevant to these functions, but the structural basis for this activity is poorly understood. In this work, we define a minimal RNA polymerase interaction domain in PcrA, and report its crystal structure at 1.5 Å resolution. The domain adopts a Tudor-like fold that is similar to other RNA polymerase interaction domains, including that of the prototype transcription-repair coupling factor Mfd. Removal or mutation of the interaction domain reduces the ability of PcrA/UvrD to interact with and to remodel RNA polymerase complexes in vitro. The implications of this work for our understanding of the role of PcrA/UvrD at the interface of DNA replication, transcription and repair are discussed.

Inhibiting translation elongation can aid genome duplication in Escherichia coli.

Conflicts between replication and transcription challenge chromosome duplication. Escherichia coli replisome movement along transcribed DNA is promoted by Rep and UvrD accessory helicases with Δrep ΔuvrD cells being inviable under rapid growth conditions. We have discovered that mutations in a tRNA gene, aspT, in an aminoacyl tRNA synthetase, AspRS, and in a translation factor needed for efficient proline-proline bond formation, EF-P, suppress Δrep ΔuvrD lethality. Thus replication-transcription conflicts can be alleviated by the partial sacrifice of a mechanism that reduces replicative barriers, namely translating ribosomes that reduce RNA polymerase backtracking. Suppression depends on RelA-directed synthesis of (p)ppGpp, a signalling molecule that reduces replication-transcription conflicts, with RelA activation requiring ribosomal pausing. Levels of (p)ppGpp in these suppressors also correlate inversely with the need for Rho activity, an RNA translocase that can bind to emerging transcripts and displace transcription complexes. These data illustrate the fine balance between different mechanisms in facilitating gene expression and genome duplication and demonstrate that accessory helicases are a major determinant of this balance. This balance is also critical for other aspects of bacterial survival: the mutations identified here increase persistence indicating that similar mutations could arise in naturally occurring bacterial populations facing antibiotic challenge.

Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones.

Our previous paper (Wilkinson et al, 2016) used high-resolution cryo-electron microscopy to solve the structure of the Escherichia coli RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in E. coli or Klebsiella pneumoniae increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in K. pneumoniae clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy.

Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination.

In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition.

Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation.

The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed.

Probing DNA helicase kinetics with temperature-controlled magnetic tweezers.

Motor protein functions like adenosine triphosphate (ATP) hydrolysis or translocation along molecular substrates take place at nanometric scales and consequently depend on the amount of available thermal energy. The associated rates can hence be investigated by actively varying the temperature conditions. In this article, a thermally controlled magnetic tweezers (MT) system for single-molecule experiments at up to 40 °C is presented. Its compact thermostat module yields a precision of 0.1 °C and can in principle be tailored to any other surface-coupled microscopy technique, such as tethered particle motion (TPM), nanopore-based sensing of biomolecules, or super-resolution fluorescence imaging. The instrument is used to examine the temperature dependence of translocation along double-stranded (ds)DNA by individual copies of the protein complex AddAB, a helicase-nuclease motor involved in dsDNA break repair. Despite moderately lower mean velocities measured at sub-saturating ATP concentrations, almost identical estimates of the enzymatic reaction barrier (around 21-24 k(B)T) are obtained by comparing results from MT and stopped-flow bulk assays. Single-molecule rates approach ensemble values at optimized chemical energy conditions near the motor, which can withstand opposing loads of up to 14 piconewtons (pN). Having proven its reliability, the temperature-controlled MT described herein will eventually represent a routinely applied method within the toolbox for nano-biotechnology.

Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus.

Single-stranded DNA-binding protein (SSB) is a well characterized ubiquitous and essential bacterial protein involved in almost all aspects of DNA metabolism. Using the Bacillus subtilis SSB we have generated a reagentless SSB biosensor that can be used as a helicase probe in B. subtilis and closely related gram positive bacteria. We have demonstrated the utility of the probe in a DNA unwinding reaction using a helicase from Bacillus and for the first time, characterized the B. subtilis SSB's DNA binding mode switching and stoichiometry. The importance of SSB in DNA metabolism is not limited to simply binding and protecting ssDNA during DNA replication, as previously thought. It interacts with an array of partner proteins to coordinate many different aspects of DNA metabolism. In most cases its interactions with partner proteins is species-specific and for this reason, knowing how to produce and use cognate reagentless SSB biosensors in different bacteria is critical. Here we explain how to produce a B. subtilis SSB probe that exhibits 9-fold fluorescence increase upon binding to single stranded DNA and can be used in all related gram positive firmicutes which employ drastically different DNA replication and repair systems than the widely studied Escherichia coli. The materials to produce the B. subtilis SSB probe are commercially available, so the methodology described here is widely available unlike previously published methods for the E. coli SSB.