inducing-site-specific-replication-blockage-e-coli-using-fluorescent

Inducing a Site Specific Replication Blockage in <i>E. coli</i> Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

Flp and Cre-mediated recombination on symmetrized FRT and loxP sites, respectively, in circular plasmid substrates yield both DNA inversion and deletion. However, upon sequestering three negative supercoils outside the recombination complex using the resII-resIII synapse formed by Tn3 resolvase and the LER synapse formed by phage Mu transposase in the case of Flp and Cre, respectively, the reactions are channeled towards inversion at the expense of deletion. The inversion product is a trefoil, its unique topology being conferred by the external resolvase or LER synapse. Thus, Flp and Cre assign their symmetrized substrates a strictly antiparallel orientation with respect to strand cleavage and exchange. These conclusions are supported by the product profiles from tethered parallel and antiparallel native FRT sites in dilution and competition assays. Furthermore, the observed recombination bias favoring deletion over inversion in a nicked circular substrate containing two symmetrized FRT sites is consistent with the predictions from Monte Carlo simulations based on antiparallel synapsis of the DNA partners.

Symmetric DNA sites are functionally asymmetric within Flp and Cre site-specific DNA recombination synapses.

The eukaryotic pre-replication complex is assembled at replication origins in a reaction called licensing. Licensing involves the interactions of a variety of proteins including the origin recognition complex (ORC), Cdc6 and the Mcm2-7 helicase, homologues of which are also found in archaea. The euryarchaeote Archaeoglobus fulgidus encodes two genes with homology to Orc/Cdc6 and a single Mcm homologue. The A.fulgidus Mcm protein and one Orc/Cdc6 homologue have been purified and investigated in vitro. The Mcm protein is an ATP-dependent, hexameric helicase that can unwind between 200 and 400 bp of duplex DNA. Deletion of 112 amino acids from the N-terminus of A.f Mcm produced a protein, which was still capable of forming a hexamer, was competent in DNA binding and was able to unwind at least 1 kb of duplex DNA. The purified Orc/Cdc6 homologue was also able to bind DNA. Both Mcm and Orc/Cdc6 show a preference for specific DNA structures, namely molecules containing a single stranded bubble that mimics early replication intermediates. Nuclease protection showed that the binding sites for Mcm and Orc/Cdc6 overlap. The Orc/Cdc6 protein bound more tightly to these substrates and was able to displace pre-bound Mcm hexamer.

Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus.

Archaea contain one or more proteins with homology to eukaryotic ORC/Cdc6 proteins. Sequence analysis suggests the existence of at least two subfamilies of these proteins, for which we propose the nomenclature ORC1 and ORC2. We have determined crystal structures of the ORC2 protein from the archaeon Aeropyrum pernix in complexes with ADP or a non-hydrolysable ATP analogue, ADPNP. Between two crystal forms, there are three crystallographically independent views of the ADP complex and two of the ADPNP complex. The protein molecules in the three complexes with ADP adopt very different conformations, while the two complexes with ADPNP are the same. These structures indicate that there is considerable conformational flexibility in ORC2 but that ATP binding stabilises a single conformation. We show that the ORC2 protein can bind DNA, and that this activity is associated with the C-terminal domain of the protein. We present a model for the interaction of the winged helix (WH) domain of ORC2 with DNA that differs from that proposed previously for Pyrobaculum aerophilum ORC/Cdc6.

Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix.

We report an efficient, controllable, site-specific replication roadblock that blocks cell proliferation, but which can be rapidly and efficiently reversed, leading to recovery of viability. Escherichia coli replication forks of both polarities stalled in vivo within the first 500 bp of a 10 kb repressor-bound array of operator DNA-binding sites. Controlled release of repressor binding led to rapid restart of the blocked replication fork without the participation of homologous recombination. Cytological tracking of fork stalling and restart showed that the replisome-associated SSB protein remains associated with the blocked fork for extended periods and that duplication of the fluorescent foci associated with the blocked operator array occurs immediately after restart, thereby demonstrating a lack of sister cohesion in the region of the array. Roadblocks positioned near oriC or the dif site did not prevent replication and segregation of the rest of the chromosome.

Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo.

We have characterised the interaction of the Aeropyrum pernix origin recognition complex proteins (ORC1 and ORC2) with DNA using DNase I footprinting. Each protein binds upstream of its respective gene. However, ORC1 protein alone interacts more tightly with an additional region containing multiple origin recognition box (ORB) sites that we show to be a replication origin. At this origin, there are four ORB elements disposed either side of an A+T-rich region. An ORC1 protein dimer binds at each of these ORB sites. Once all four ORB sites have bound ORC1 protein, there is a transition to a higher-order assembly with a defined alteration in topology and superhelicity. Furthermore, after this transition, the A+T-rich region becomes sensitive to digestion by DNase I and P1 nuclease, revealing that the transition promotes distortion of the DNA in this region, presumably as a prelude to loading of MCM helicase.

Biochemical analysis of a DNA replication origin in the archaeon Aeropyrum pernix.

A challenge for chromosome segregation in all domains of life is the formation of catenated progeny chromosomes, which arise during replication as a consequence of the interwound strands of the DNA double helix. Topoisomerases play a key role in DNA unlinking both during and at the completion of replication. Here we report that chromosome unlinking can instead be accomplished by multiple rounds of site-specific recombination. We show that step-wise, site-specific recombination by XerCD-dif or Cre-loxP can unlink bacterial chromosomes in vivo, in reactions that require KOPS-guided DNA translocation by FtsK. Furthermore, we show that overexpression of a cytoplasmic FtsK derivative is sufficient to allow chromosome unlinking by XerCD-dif recombination when either subunit of TopoIV is inactivated. We conclude that FtsK acts in vivo to simplify chromosomal topology as Xer recombination interconverts monomeric and dimeric chromosomes.

Unlinking chromosome catenanes in vivo by site-specific recombination.

Dimeric circular chromosomes, formed by recombination between monomer sisters, cannot be segregated to daughter cells at cell division. XerCD site-specific recombination at the Escherichia coli dif site converts these dimers to monomers in a reaction that requires the DNA translocase FtsK. Short DNA sequences, KOPS (GGGNAGGG), which are polarized toward dif in the chromosome, direct FtsK translocation. FtsK interacts with KOPS through a C-terminal winged helix domain gamma. The crystal structure of three FtsKgamma domains bound to 8 bp KOPS DNA demonstrates how three gamma domains recognize KOPS. Using covalently linked dimers of FtsK, we infer that three gamma domains per hexamer are sufficient to recognize KOPS and load FtsK and subsequently activate recombination at dif. During translocation, FtsK fails to recognize an inverted KOPS sequence. Therefore, we propose that KOPS act solely as a loading site for FtsK, resulting in a unidirectionally oriented hexameric motor upon DNA.

Molecular mechanism of sequence-directed DNA loading and translocation by FtsK.

Sporulation in Bacillus subtilis requires asymmetric cell division, chromosome transfer into the spore and establishment of differential gene expression patterns. Several recent studies highlight the key roles of the SpoIIIE motor in this process.

Sporulation: SpoIIIE is the key to cell differentiation.

Replication in archaea is carried out by proteins that are homologues of eukaryotic counterparts. However, the archaeal systems tend to be much simpler with fewer different genes encoding the core functions than in eukaryotic counterparts. In many archaea, there is a single minichromosome maintenance (MCM) homologue, presumed to be the replicative helicase and between one and three origin recognition complex (ORC) homologues involved in binding to the replication origins. Here we describe the cloning and characterization of the MCM protein from the crenarchaeote Aeropyrum pernix. Like other eukaryotic and archaeal MCM proteins, it is found to be an ATP-dependent DNA helicase, and the putative active site residues involved in ATP binding and hydrolysis are confirmed by mutation. Deletion of the N-terminal 256 amino acids yielded a protein with higher ATPase activity in the absence of DNA and retained robust helicase activity. Interactions with the ORC proteins of A. pernix were examined, and it was found that both ORC homologues could inhibit the helicase activity of MCM. Further it was found that ORC2 could autophosphorylate in the presence of ATP and more remarkably could phosphorylate MCM in a species-specific manner.

Biochemical characterization of the minichromosome maintenance (MCM) protein of the crenarchaeote Aeropyrum pernix and its interactions with the origin recognition complex (ORC) proteins.

Escherichia coli FtsK is a septum-located DNA translocase that co-ordinates the late stages of cytokinesis and chromosome segregation. Relatives of FtsK are present in most bacteria; in Bacillus subtilis, the FtsK orthologue, SpoIIIE, transfers the majority of a chromosome into the forespore during sporulation. DNA translocase activity is contained within a ~ 512-amino-acid C-terminal domain, which is divided into three subdomains: alpha, beta and gamma. alpha and beta comprise the translocation motor, and gamma is a regulatory domain that interacts with DNA and with the XerD recombinase. In vitro rates of translocation of ~ 5 kb.s(-1) have been measured for both FtsK and SpoIIIE, whereas, in vivo, SpoIIIE has a comparable rate of translocation. Translocation by both of these proteins is not only rapid, but also directed by DNA sequence. This directionality requires interaction of the gamma subdomain with specific 8 bp DNA asymmetric sequences that are oriented co-directionally with replication direction of the bacterial chromosome. The gamma subdomain also interacts with the XerCD site-specific recombinase to activate chromosome unlinking by recombination at the chromosomal dif site. In the present paper, the properties in vivo and in vitro of FtsK and its relatives are discussed in relation to the biological functions of these remarkable enzymes.

The Escherichia coli DNA translocase FtsK.

FtsK translocates dsDNA directionally at >5 kb/s, even under strong forces. In vivo, the action of FtsK at the bacterial division septum is required to complete the final stages of chromosome unlinking and segregation. Despite the availability of translocase structures, the mechanism by which ATP hydrolysis is coupled to DNA translocation is not understood. Here, we use covalently linked translocase subunits to gain insight into the DNA translocation mechanism. Covalent trimers of wild-type subunits dimerized efficiently to form hexamers with high translocation activity and an ability to activate XerCD-dif chromosome unlinking. Covalent trimers with a catalytic mutation in the central subunit formed hexamers with two mutated subunits that had robust ATPase activity. They showed wild-type translocation velocity in single-molecule experiments, activated translocation-dependent chromosome unlinking, but had an impaired ability to displace either a triplex oligonucleotide, or streptavidin linked to biotin-DNA, during translocation along DNA. This separation of translocation velocity and ability to displace roadblocks is more consistent with a sequential escort mechanism than stochastic, hand-off, or concerted mechanisms.

Separating speed and ability to displace roadblocks during DNA translocation by FtsK.

FtsK is a double-stranded DNA translocase, a motor that converts the chemical energy of binding and hydrolysing ATP into movement of a DNA substrate. It moves DNA at an amazing rate->5000 bp per second-and is powerful enough to remove other proteins from the DNA. In bacteria it is localised to the site of cell division, the septum, where it functions as a DNA pump at the late stages of the cell cycle, to expedite cytokinesis and chromosome segregation. The N terminus of the protein is involved in the cell-cycle-specific localisation and assembly of the cell-division machinery, whereas the C terminus forms the motor. The motor portion of FtsK has been studied by a combination of biochemistry, genetics, X-ray crystallography and single-molecule mechanical assays, and these will be the focus here. The motor can be divided into three subdomains: α, β and γ. The α and β domains multimerise to produce a hexameric ring with a central channel for dsDNA, and contain a RecA-like nucleotide-binding/hydrolysis fold. The motor is given directionality by the regulatory γ domain, which binds to polarised chromosomal sequences-5'-GGGNAGGG-3', known as KOPS-to ensure that the motor is loaded onto DNA in a specific orientation such that subsequent translocation is always towards the region of the chromosome where replication usually terminates (the terminus), and specifically to the 28 bp dif site, located in this region. Once the FtsK translocase has located the dif site it then interacts with the XerCD site-specific recombinases to activate recombination.

FtsK DNA translocase: the fast motor that knows where it's going.

FtsK is a multifunctional, multidomain protein that acts to co-ordinate chromosome unlinking, segregation and cell division. In this issue of Molecular Microbiology, the report by Dubarry et al. reveals new insight into the surprisingly complex relationship between the different activities of FtsK. The new study makes extensive use of fusion proteins to highlight the role of the FtsK 'linker' domain in helping to co-ordinate these processes. This, taken together with previous studies, suggests that FtsK is intimately involved in septum constriction, physically contacting several other divisome proteins. Further, it is attractive to speculate that FtsK can regulate the late stages of septation to act as a checkpoint to ensure DNA is fully cleared from the septum before it is allowed to close, as well as being the driving force to unlink the chromosomes and segregate the DNA away from the septum.

FtsK--a bacterial cell division checkpoint?

The FtsK translocase pumps dsDNA directionally at ∼5 kb/s and facilitates chromosome unlinking by activating XerCD site-specific recombination at dif, located in the replication terminus of the Escherichia coli chromosome. We show directly that the γ regulatory subdomain of FtsK activates XerD catalytic activity to generate Holliday junction intermediates that can then be resolved by XerC. Furthermore, we demonstrate that γ can activate XerCD-dif recombination in the absence of the translocase domain, when it is fused to XerCD, or added in isolation. In these cases the recombination products are topologically complex and would impair chromosome unlinking. We propose that FtsK translocation and activation of unlinking are normally coupled, with the translocation being essential for ensuring that the products of recombination are topologically unlinked, an essential feature of the role of FtsK in chromosome segregation.

Activation of XerCD-dif recombination by the FtsK DNA translocase.

FtsK is a multifunctional protein, which, in Escherichia coli, co-ordinates the essential functions of cell division, DNA unlinking and chromosome segregation. Its C-terminus is a DNA translocase, the fastest yet characterized, which acts as a septum-localized DNA pump. FtsK's C-terminus also interacts with the XerCD site-specific recombinases which act at the dif site, located in the terminus region. The motor domain of FtsK is an active translocase in vitro, and, when incubated with XerCD and a supercoiled plasmid containing two dif sites, recombination occurs to give unlinked circular products. Despite years of research the mechanism for this novel form of topological filter remains unknown.

Simple topology: FtsK-directed recombination at the dif site.

In Escherichia coli, complete unlinking of newly replicated sister chromosomes is required to ensure their proper segregation at cell division. Whereas replication links are removed primarily by topoisomerase IV, XerC/XerD-dif site-specific recombination can mediate sister chromosome unlinking in Topoisomerase IV-deficient cells. This reaction is activated at the division septum by the DNA translocase FtsK, which coordinates the last stages of chromosome segregation with cell division. It has been proposed that, after being activated by FtsK, XerC/XerD-dif recombination removes DNA links in a stepwise manner. Here, we provide a mathematically rigorous characterization of this topological mechanism of DNA unlinking. We show that stepwise unlinking is the only possible pathway that strictly reduces the complexity of the substrates at each step. Finally, we propose a topological mechanism for this unlinking reaction.

FtsK-dependent XerCD-dif recombination unlinks replication catenanes in a stepwise manner.

Biomotors are extensively involved in biological processes including cell mitosis, bacterial binary fission, DNA replication, DNA repair, homologous recombination, Holliday junction resolution, RNA transcription, and viral genome packaging. Traditionally, they were classified into two categories including linear and rotation motors. In 2013, a third class of motor by revolution mechanism without rotation was discovered. In this issue of "Structure and mechanisms of nanomotors in the cells", four comprehensive reviews are published to address the latest advancements of the structure and motion mechanism of a variety of biomotors in archaea, animal viruses, bacteria, and bacteriophages.

Two classes of nucleic acid translocation motors: rotation and revolution without rotation.

Replication of chromosomal DNA must be carried out to completion in order for a cell to proliferate. However, replication forks can stall during this process for a variety of reasons, including nucleoprotein 'roadblocks' and DNA lesions. In these circumstances the replisome copying the DNA may disengage from the chromosome to allow various repair processes to restore DNA integrity and enable replication to continue. Here, we report the in vivo stability of the replication fork when it encounters a nucleoprotein blockage in Escherichia coli. Using a site-specific and reversible protein block system in conjunction with the temperature sensitive DnaC helicase loader and DnaB replicative helicase, we monitored the disappearance of the Y-shaped DNA replication fork structures using neutral-neutral 2D agarose gels. We show the replication fork collapses within 5 min of encountering the roadblock. Therefore, the stalled replication fork does not pause at a block in a stable confirmation for an extended period of time as previously postulated.

Stability of blocked replication forks in vivo.

The ubiquitous biological nanomotors were classified into two categories in the past: linear and rotation motors. In 2013, a third type of biomotor, revolution without rotation (http://rnanano.osu.edu/movie.html), was discovered and found to be widespread among bacteria, eukaryotic viruses, and double-stranded DNA (dsDNA) bacteriophages. This review focuses on recent findings about various aspects of motors, including chirality, stoichiometry, channel size, entropy, conformational change, and energy usage rate, in a variety of well-studied motors, including FoF1 ATPase, helicases, viral dsDNA-packaging motors, bacterial chromosome translocases, myosin, kinesin, and dynein. In particular, dsDNA translocases are used to illustrate how these features relate to the motion mechanism and how nature elegantly evolved a revolution mechanism to avoid coiling and tangling during lengthy dsDNA genome transportation in cell division. Motor chirality and channel size are two factors that distinguish rotation motors from revolution motors. Rotation motors use right-handed channels to drive the right-handed dsDNA, similar to the way a nut drives the bolt with threads in same orientation; revolution motors use left-handed motor channels to revolve the right-handed dsDNA. Rotation motors use small channels (<2 nm in diameter) for the close contact of the channel wall with single-stranded DNA (ssDNA) or the 2-nm dsDNA bolt; revolution motors use larger channels (>3 nm) with room for the bolt to revolve. Binding and hydrolysis of ATP are linked to different conformational entropy changes in the motor that lead to altered affinity for the substrate and allow work to be done, for example, helicase unwinding of DNA or translocase directional movement of DNA.

Article	Year
Symmetric DNA sites are functionally asymmetric within Flp and Cre site-specific DNA recombination synapses. Journal of molecular biology\| PubMed ID: 12096907	2002
Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus. Nucleic acids research\| PubMed ID: 12907732	2003
Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix. Journal of molecular biology\| PubMed ID: 15465044	2004
Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo. The EMBO journal\| PubMed ID: 16724111	2006
Biochemical analysis of a DNA replication origin in the archaeon Aeropyrum pernix. Journal of molecular biology\| PubMed ID: 16978641	2006
Unlinking chromosome catenanes in vivo by site-specific recombination. The EMBO journal\| PubMed ID: 17805344	2007
Molecular mechanism of sequence-directed DNA loading and translocation by FtsK. Molecular cell\| PubMed ID: 18722176	2008
Sporulation: SpoIIIE is the key to cell differentiation. Current biology : CB\| PubMed ID: 18812085	2008
Biochemical characterization of the minichromosome maintenance (MCM) protein of the crenarchaeote Aeropyrum pernix and its interactions with the origin recognition complex (ORC) proteins. Biochemistry\| PubMed ID: 19053250	2008
The Escherichia coli DNA translocase FtsK. Biochemical Society transactions\| PubMed ID: 20298190	2010
Separating speed and ability to displace roadblocks during DNA translocation by FtsK. The EMBO journal\| PubMed ID: 20379135	2010
FtsK DNA translocase: the fast motor that knows where it's going. Chembiochem : a European journal of chemical biology\| PubMed ID: 20922738	2010
FtsK--a bacterial cell division checkpoint? Molecular microbiology\| PubMed ID: 21155139	2010
Activation of XerCD-dif recombination by the FtsK DNA translocase. Nucleic acids research\| PubMed ID: 21371996	2011
Simple topology: FtsK-directed recombination at the dif site. Biochemical Society transactions\| PubMed ID: 23514160	2013
FtsK-dependent XerCD-dif recombination unlinks replication catenanes in a stepwise manner. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24218579	2013
Two classes of nucleic acid translocation motors: rotation and revolution without rotation. Cell & bioscience\| PubMed ID: 25276341	2014
Stability of blocked replication forks in vivo. Nucleic acids research\| PubMed ID: 26490956	2016
Biological Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism. Microbiology and molecular biology reviews : MMBR\| PubMed ID: 26819321	2016

Ian Grainge

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