Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.

Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue.

Mechanism of transcription modulation by the transcription-repair coupling factor.

Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

Resolving the Gordian Knot: Srs2 Strips Intermediates Formed during Homologous Recombination.

Cells use a suite of specialized enzymes to repair chromosomal double-strand breaks (DSBs). Two recent studies describe how single-molecule fluorescence imaging techniques are used in the direct visualization of some of the key molecular steps involved. De Tullio et al. and Kaniecki et al. watch individual Srs2 helicase molecules disrupt repair intermediates formed by RPA, Rad51, and Rad52 on DNA during homologous recombination.

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.

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

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

Identification of Multiple Kinetic Populations of DNA-Binding Proteins in Live Cells.

Understanding how multiprotein complexes function in cells requires detailed quantitative understanding of their association and dissociation kinetics. Analysis of the heterogeneity of binding lifetimes enables the interrogation of the various intermediate states formed during the reaction. Single-molecule fluorescence imaging permits the measurement of reaction kinetics inside living organisms with minimal perturbation. However, poor photophysical properties of fluorescent probes limit the dynamic range and accuracy of measurements of off rates in live cells. Time-lapse single-molecule fluorescence imaging can partially overcome the limits of photobleaching; however, limitations of this technique remain uncharacterized. Here, we present a structured analysis of which timescales are most accessible using the time-lapse imaging approach and explore uncertainties in determining kinetic subpopulations. We demonstrate the effect of shot noise on the precision of the measurements as well as the resolution and dynamic range limits that are inherent to the method. Our work provides a convenient implementation to determine theoretical errors from measurements and to support interpretation of experimental data.

Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance.

Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow individual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA-repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA-repair processes in prokaryotes.

Design of DNA rolling-circle templates with controlled fork topology to study mechanisms of DNA replication.

Rolling-circle DNA amplification is a powerful tool employed in biotechnology to produce large from small amounts of DNA. This mode of DNA replication proceeds via a DNA topology that resembles a replication fork, thus also providing experimental access to the molecular mechanisms of DNA replication. However, conventional templates do not allow controlled access to multiple fork topologies, which is an important factor in mechanistic studies. Here we present the design and production of a rolling-circle substrate with a tunable length of both the gap and the overhang, and we show its application to the bacterial DNA-replication reaction.

Regulation of Mutagenic DNA Polymerase V Activation in Space and Time.

Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD'2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD'. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD'2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template.

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates.

How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3-0.8, 8.1, and 113-126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism.

Single-Molecule Specific Mislocalization of Red Fluorescent Proteins in Live Escherichia coli.

Tagging of individual proteins with genetically encoded fluorescent proteins (FPs) has been used extensively to study localization and interactions in live cells. Recent developments in single-molecule localization microscopy have enabled the dynamic visualization of individual tagged proteins inside living cells. However, tagging proteins with FPs is not without problems: formation of insoluble aggregates and inhibition of native functions of the protein are well-known issues. Previously reported artifacts manifest themselves at all expression levels of the FP-tagged proteins, making the design of control experiments relatively straightforward. Here, we describe a previously uncharacterized mislocalization artifact of Entacmaea quadricolor red fluorescent protein variants that is detectable at the single-molecule level in live Escherichia coli cells.

The Shu complex interacts with Rad51 through the Rad51 paralogues Rad55-Rad57 to mediate error-free recombination.

The Saccharomyces cerevisiae Shu complex, consisting of Shu1, Shu2, Csm2 and Psy3, promotes error-free homologous recombination (HR) by an unknown mechanism. Recent structural analysis of two Shu proteins, Csm2 and Psy3, has revealed that these proteins are Rad51 paralogues and mediate DNA binding of this complex. We show in vitro that the Csm2-Psy3 heterodimer preferentially binds synthetic forked DNA or 3'-DNA overhang substrates resembling structures used during HR in vivo. We find that Csm2 interacts with Rad51 and the Rad51 paralogues, the Rad55-Rad57 heterodimer and that the Shu complex functions in the same epistasis group as Rad55-Rad57. Importantly, Csm2's interaction with Rad51 is dependent on Rad55, whereas Csm2's interaction with Rad55 occurs independently of Rad51. Consistent with the Shu complex containing Rad51 paralogues, the methyl methanesulphonate sensitivity of Csm2 is exacerbated at colder temperatures. Furthermore, Csm2 and Psy3 are needed for efficient recruitment of Rad55 to DNA repair foci after DNA damage. Finally, we observe that the Shu complex preferentially promotes Rad51-dependent homologous recombination over Rad51-independent repair. Our data suggest a model in which Csm2-Psy3 recruit the Shu complex to HR substrates, where it interacts with Rad51 through Rad55-Rad57 to stimulate Rad51 filament assembly and stability, promoting error-free repair.

Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes.

Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA-D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB-DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC-DNA complex, we investigated three UvrB mutants: Y96A, a ß-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell.

Damaged DNA induced UV-damaged DNA-binding protein (UV-DDB) dimerization and its roles in chromatinized DNA repair.

UV light-induced photoproducts are recognized and removed by the nucleotide-excision repair (NER) pathway. In humans, the UV-damaged DNA-binding protein (UV-DDB) is part of a ubiquitin E3 ligase complex (DDB1-CUL4A(DDB2)) that initiates NER by recognizing damaged chromatin with concomitant ubiquitination of core histones at the lesion. We report the X-ray crystal structure of the human UV-DDB in a complex with damaged DNA and show that the N-terminal domain of DDB2 makes critical contacts with two molecules of DNA, driving N-terminal-domain folding and promoting UV-DDB dimerization. The functional significance of the dimeric UV-DDB [(DDB1-DDB2)(2)], in a complex with damaged DNA, is validated by electron microscopy, atomic force microscopy, solution biophysical, and functional analyses. We propose that the binding of UV-damaged DNA results in conformational changes in the N-terminal domain of DDB2, inducing helical folding in the context of the bound DNA and inducing dimerization as a function of nucleotide binding. The temporal and spatial interplay between domain ordering and dimerization provides an elegant molecular rationale for the unprecedented binding affinities and selectivities exhibited by UV-DDB for UV-damaged DNA. Modeling the DDB1-CUL4A(DDB2) complex according to the dimeric UV-DDB-AP24 architecture results in a mechanistically consistent alignment of the E3 ligase bound to a nucleosome harboring damaged DNA. Our findings provide unique structural and conformational insights into the molecular architecture of the DDB1-CUL4A(DDB2) E3 ligase, with significant implications for the regulation and overall organization of the proteins responsible for initiation of NER in the context of chromatin and for the consequent maintenance of genomic integrity.

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.

Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding.

Human telomeres are composed of duplex TTAGGG repeats and a 3' single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding.

Biochemical and single-molecule techniques to study accessory helicase resolution of R-loop proteins at stalled replication forks.

R-loop proteins present a stable and robust blockade to the progression of a DNA replication fork during S-phase. The consequences of this block can include mutagenesis and other irreversible chromosomal catastrophes, causing genomic instability and disease. As such, further investigation into the molecular mechanisms underlying R-loop protein resolution is warranted. The critical role of non-replicative accessory helicases in R-loop protein resolution has increasingly come into light in recent years. Such helicases include the Pif1-family, monomeric helicases that have been studied in many different contexts and that have been ascribed to a multitude of separable protective functions in the cell. In this chapter, we present protocols to study R-loop protein resolution by Pif1 helicase at stalled replication forks using purified proteins, both at the biochemical and single-molecule level. Our system uses recombinant proteins expressed in Saccharomyces cerevisiae but could apply to practically any organism of interest due to the high interspecies homology of the proteins involved in DNA replication. The methods we outline are extensible to many systems and should be applicable to studying R-loop clearance by any Superfamily (SF) 1B helicase. These techniques will further enable mechanistic research on these critical but understudied components of the genomic maintenance program.

Single-molecule studies of helicases and translocases in prokaryotic genome-maintenance pathways.

Helicases involved in genomic maintenance are a class of nucleic-acid dependent ATPases that convert the energy of ATP hydrolysis into physical work to execute irreversible steps in DNA replication, repair, and recombination. Prokaryotic helicases provide simple models to understand broadly conserved molecular mechanisms involved in manipulating nucleic acids during genome maintenance. Our understanding of the catalytic properties, mechanisms of regulation, and roles of prokaryotic helicases in DNA metabolism has been assembled through a combination of genetic, biochemical, and structural methods, further refined by single-molecule approaches. Together, these investigations have constructed a framework for understanding the mechanisms that maintain genomic integrity in cells. This review discusses recent single-molecule insights into molecular mechanisms of prokaryotic helicases and translocases.

Single-molecule imaging reveals molecular coupling between transcription and DNA repair machinery in live cells.

The Escherichia coli transcription-repair coupling factor Mfd displaces stalled RNA polymerase and delivers the stall site to the nucleotide excision repair factors UvrAB for damage detection. Whether this handoff from RNA polymerase to UvrA occurs via the Mfd-UvrA2-UvrB complex or alternate reaction intermediates in cells remains unclear. Here, we visualise Mfd in actively growing cells and determine the catalytic requirements for faithful recruitment of nucleotide excision repair proteins. We find that ATP hydrolysis by UvrA governs formation and disassembly of the Mfd-UvrA2 complex. Further, Mfd-UvrA2-UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition are impaired in successful handoff. Our single-molecule dissection of interactions of Mfd with its partner proteins inside live cells shows that the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner.

Single-molecule live-cell imaging visualizes parallel pathways of prokaryotic nucleotide excision repair.

In the model organism Escherichia coli, helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled by lesions. Ultimately, damage recognition is mediated by UvrA, followed by verification by UvrB. Here we characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB by following functional, fluorescently tagged UvrA molecules in live TCR-deficient or wild-type cells. The lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, the lifetimes of UvrA strongly depended on, and matched those of Mfd. Overall, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.