Search

CRISPR-Cas immunity protects prokaryotes against invading genetic elements.sup.1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers).sup.2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear.sup.6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms--including single-stranded DNA and partial duplexes--in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci. Cas1-Cas2 selects precursor prespacers from DNA fragments in a length- and PAM-sequence-dependent manner, and these precursors are trimmed by DnaQ exonucleases to enable integration into the CRISPR locus in the correct orientation., Author(s): Sungchul Kim [sup.1] , Luuk Loeff [sup.1] , Sabina Colombo [sup.1] , Slobodan Jergic [sup.2] [sup.3] , Stan J. J. Brouns [sup.1] , Chirlmin Joo [sup.1] Author Affiliations: (1) [...]

In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites (1). Escherichia coli sets a 'replication fork trap' that allows the first arriving fork to enter but not to leave the terminus region (2-5). The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction (3-7). However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro (8, 9). Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes., In the circular E. coli chromosome, two replication forks move from the replication origin to converge opposite in a region that contains ten 23-base-pair Ter (termination) sites and the dif [...]

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the Escherichia coli replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes. [ABSTRACT FROM AUTHOR]

High-field pulse electron paramagnetic resonance (EPR) is used to introduce [Gd.sup.3+] spin labeling for nanometer-range distance measurements in proteins. The results demonstrated the application of [Gd.sup.3+] labeling as a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels.

A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding camp, at a single site on the diner. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to Beta(sub 2) in various stages or circumstances of DNA replication and repair.

Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. InEscherichia coli, a “replication fork trap” – found within a chromosomal region where forks are allowed to enter but not leave – is set by the protein–DNA roadblock Tus–Ter. The exact sequence of events by which Tus–Terblocks replisomes approaching from one direction but not the other has been the subject of controversy for many decades. Specific protein–protein interactions between the nonpermissive face of Tus and the approaching helicase were challenged by biochemical and structural studies. These studies show that it is the helicase-induced strand separation that triggers the formation of new Tus–Terinteractions at the nonpermissive face – interactions that result in a highly stable “locked” complex. This controversy recently gained renewed attention as three single-molecule-based studies scrutinized this elusive Tus–Termechanism – leading to new findings and refinement of existing models, but also generating new questions. Here, we discuss and compare the findings of each of the single-molecule studies to find their common ground, pinpoint the crucial differences that remain, and push the understanding of this bipartite DNA–protein system further. [ABSTRACT FROM PUBLISHER]

The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation. [ABSTRACT FROM AUTHOR]

Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E. coli β clamp in real time by using single-molecule FRET (smFRET). Three successive FRET states were retained for 0.3 s, 0.7 s, and 9 min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3 s, and after 0.7 s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9 min. An additional single-molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single-molecule polarization and FRET studies thus revealed the real-time dynamics of the ATP-hydrolysis-dependent 3D conformational change of the β clamp during loading at a ss/dsDNA junction. [ABSTRACT FROM AUTHOR]

Processive DNA synthesis by the αɛθ core of the Escherichia coli Pol III replicase requires it to be bound to the β2 clamp via a site in the α polymerase subunit. How the ɛ proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of ɛ. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in ɛ that, in conjunction with the site in α, maintains a closed state of the αɛθ-β2 replicase in the polymerization mode of DNA synthesis. The ɛ-β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states. [ABSTRACT FROM AUTHOR]

During DNA replication, repetitive synthesis of discrete Okazaki fragments requires mechanisms that guarantee DNA polymerase, clamp, and primase proteins are present for every cycle. In Escherichia coli, this process proceeds through transfer of the lagging-strand polymerase from the β sliding clamp left at a completed Okazaki fragment to a clamp assembled on a new RNA primer. These lagging-strand clamps are thought to be bound by the replisome from solution and loaded a new for every fragment. Here, we discuss a surprising, alternative lagging-strand synthesis mechanism: efficient replication in the absence of any clamps other than those assembled with the replisome. Using single-molecule experiments, we show that replication complexes pre-assembled on DNA support synthesis of multiple Okazaki fragments in the absence of excess β clamps. The processivity of these replisomes, but not the number of synthesized Okazaki fragments, is dependent on the frequency of RNA-primer synthesis. These results broaden our understanding of lagging-strand synthesis and emphasize the stability of the replisome to continue synthesis without new clamps. [ABSTRACT FROM AUTHOR]

Summary: Multi-protein clamp loader complexes are required to load sliding clamps onto DNA. In Escherichia coli the clamp loader contains three DnaX (τ/γ) proteins, δ, and δ′, which together form an asymmetric pentameric ring that also interacts with ψχ. Here we used mass spectrometry to examine the assembly and dynamics of the clamp loader complex. We find that γ exists exclusively as a stable homotetramer, while τ is in a monomer-dimer-trimer-tetramer equilibrium. δ′ plays a direct role in the assembly as a τ/γ oligomer breaker, thereby facilitating incorporation of lower oligomers. With δ′, both δ and ψχ stabilize the trimeric form of DnaX, thus completing the assembly. When τ and γ are present simultaneously, mimicking the situation in vivo, subunit exchange between τ and γ tetramers occurs rapidly to form heterocomplexes but is retarded when δδ′ is present. The implications for intracellular assembly of the DNA polymerase III holoenzyme are discussed. [Copyright &y& Elsevier]

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcuspneumoniae n-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN[supK252 + 255A], SEN[supK344A], SEN[supK334A], SEN[supK344E], SEN[supK435L, and SEN[supΔ434-435]) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN[supK344E], as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN[supK3444E] had lost a-enolase activity, which was also reduced in SEN[supK334A] and SEN[supΔ434-435]. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN[supK252 + 255A], SEN[supK435L], and SEN[supΔ434-435]. The lysine residues at positions 252, 255,434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures. [ABSTRACT FROM AUTHOR]

We present single-molecule studies of the Escherichia coli replication machinery. We visualize individual E. coli DNA polymerase III (Pol III) holoenzymes engaging in primer extension and leading-strand synthesis. When coupled to the replicative helicase DnaB, Pol III mediates leading-strand synthesis with a processivity of 10.5 kilobases (kb), eight-fold higher than that by Pol III alone. Addition of the primase DnaG causes a three-fold reduction in the processivity of leading-strand synthesis, an effect dependent upon the DnaB-DnaG protein-protein interaction rather than primase activity. A single-molecule analysis of the replication kinetics with varying DnaG concentrations indicates that a cooperative binding of two or three DnaG monomers to DnaB halts synthesis. Modulation of DnaB helicase activity through the interaction with DnaG suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during slow primer synthesis on the lagging strand. [ABSTRACT FROM AUTHOR]

The efficiency of cell-free protein synthesis combined with combinatorial selective 15N-labelling provides a method for the rapid assignment of 15N-HSQC cross-peaks to the 19 different non-proline amino-acid types from five 15N-HSQC spectra. This strategy was explored with two different constructs of the C-terminal domain V of the τ subunit of the Escherichia coli DNA polymerase III holoenzyme, τC16 and τC14. Since each of the five 15N-HSQC spectra contained only about one third of the cross-peaks present in uniformly labelled samples, spectral overlap was much reduced. All 15N-HSQC cross-peaks of the backbone amides could be assigned to the correct amino-acid type. Availability of the residue-type information greatly assisted the evaluation of the changes in chemical shifts observed for corresponding residues in τC16 vs. those in τC14, and the analysis of the structure and mobility of the C-terminal residues present in τC16 but not in τC14. [ABSTRACT FROM AUTHOR]

Cell-free protein synthesis systems provide facile access to proteins in a nascent state that enables formation of soluble, native protein–protein complexes even if one of the protein components is prone to self-aggregation and precipitation. Combined with selective isotope-labeling, this allows the rapid analysis of protein–protein interactions with few 15N-HSQC spectra. The concept is demonstrated with binary and ternary complexes between the χ, ψ and γ subunits of Escherichia coli DNA polymerase III: nascent, selectively 15N-labeled ψ produced in the presence of χ resulted in a soluble, correctly folded χ-ψ complex, whereas ψ alone precipitated irrespective of whether γ was present or not. The 15N-HSQC spectra showed that the N-terminal segment of ψ is mobile in the χ-ψ complex, yet important for its binding to γ. The sample preparation was greatly enhanced by an autoinduction strategy, where the T7 RNA polymerase needed for transcription of a gene in a T7-promoter vector was produced in situ. [ABSTRACT FROM AUTHOR]

Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity. There are at least two functional modes of interaction of Pol III and DnaB on DNA The interaction between the CLC and DnaB strengthens ∼500-fold upon DnaG binding A single copy of the τ subunit in the CLC is responsible for binding DnaB Multiple Pol IIIs can be at the fork, subject to the status of DnaB-DnaG interaction Monachino et al. show that dynamic interaction of the Escherichia coli DnaG primase and DnaB helicase affects the stability of the replisome and the cycling of DNA polymerase III complexes at the replication fork through a conformational switch in DnaB that toggles its affinity for the polymerase. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Methods for measuring nanometer-scale distances between specific sites in proteins are essential for analysis of their structure and function. In this work we introduce Gd3+ spin labeling for nanometer- range distance measurements in proteins by high-field pulse electron paramagnetic resonance (EPR). To evaluate the performance of such measurements, we carried out four-pulse double-electron electron resonance (DEER) measurements on two proteins, p75ICD and ΤC4, labeled at strategically selected sites with either two nitroxides or two Gd3+ spin labels. In analogy to conventional site-directed spin labeling using nitroxides, Gd3+ tags that are derivatives of dipicolinic acid were covalently attached to cysteine thiol groups. Measurements were carried out on X-band (~9.5 GHz, 0.35 T) and W-band (95 GHz, 3.5 T) spectrometers for the nitroxide-labeled proteins and at W-band for the Gd3+-labeled proteins. In the protein p75ICD, the orientations of the two nitroxides were found to be practically uncorrelated, and therefore the distance distribution could as readily be obtained at W-band as at X-band. The measured Gd3+-Gd3+ distance distribution had a maximum at 2.9 nm, as compared to 2.5 nm for the nitroxides. In the protein rcl4, however, the orientations of the nitroxides were correlated, and the W-band measurements exhibited strong orientation selection that prevented a straightforward extraction of the distance distribution. The X-band measurements gave a nitroxide-nitroxide distance distribution with a maximum at 2.5 nm, and the W-band measurements gave a Gd3+-Gd3+ distance distribution with a maximum at 3.4 nm. The Gd3+-Gd3+ distance distributions obtained are in good agreement with expectations from structural models that take into account the flexibility of the tags and their tethers to the cysteine residues. These results show that Gd3+ labeling is a viable technique for distance measurements at high fields that features an order of magnitude sensitivity improvement, in terms of protein quantity, over X-band pulse EPR measurements using nitroxide spin labels. Its advantage over W-band distance measurements using nitroxides stems from an intrinsic absence of orientation selection. [ABSTRACT FROM AUTHOR]