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1.  A bipolar DNA helicase gene, herA, clusters with rad50, mre11 and nurA genes in thermophilic archaea 
Nucleic Acids Research  2004;32(4):1439-1447.
We showed previously that rad50 and mre11 genes of thermophilic archaea are organized in an operon-like structure with a third gene (nurA) encoding a 5′ to 3′ exonuclease. Here, we show that the rad50, mre11 and nurA genes from the hyperthermo philic archaeon Sulfolobus acidocaldarius are co-transcribed with a fourth gene encoding a DNA helicase. This enzyme (HerA) is the prototype of a new class of DNA helicases able to utilize either 3′ or 5′ single-stranded DNA extensions for loading and subsequent DNA duplex unwinding. To our knowledge, DNA helicases capable of translocating along the DNA in both directions have not been identified previously. Sequence analysis of HerA shows that it is a member of the TrwB, FtsK and VirB4/VirD4 families of the PilT class NTPases. HerA homologs are found in all thermophilic archaeal species and, in all cases except one, the rad50, mre11, nurA and herA genes are grouped together. These results suggest that the archaeal Rad50–Mre11 complex might act in association with a 5′ to 3′ exonuclease (NurA) and a bipolar DNA helicase (HerA) indicating a probable involvement in the initiation step of homologous recombination.
doi:10.1093/nar/gkh283
PMCID: PMC390275  PMID: 14990749
2.  The Mre11 protein interacts with both Rad50 and the HerA bipolar helicase and is recruited to DNA following gamma irradiation in the archaeon Sulfolobus acidocaldarius 
Background
The ubiquitous Rad50 and Mre11 proteins play a key role in many processes involved in the maintenance of genome integrity in Bacteria and Eucarya, but their function in the Archaea is presently unknown. We showed previously that in most hyperthermophilic archaea, rad50-mre11 genes are linked to nurA encoding both a single-strand endonuclease and a 5' to 3' exonuclease, and herA, encoding a bipolar DNA helicase which suggests the involvement of the four proteins in common molecular pathway(s). Since genetic tools for hyperthermophilic archaea are just emerging, we utilized immuno-detection approaches to get the first in vivo data on the role(s) of these proteins in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius.
Results
We first showed that S. acidocaldarius can repair DNA damage induced by high doses of gamma rays, and we performed a time course analysis of the total levels and sub-cellular partitioning of Rad50, Mre11, HerA and NurA along with the RadA recombinase in both control and irradiated cells. We found that during the exponential phase, all proteins are synthesized and display constant levels, but that all of them exhibit a different sub-cellular partitioning. Following gamma irradiation, both Mre11 and RadA are immediately recruited to DNA and remain DNA-bound in the course of DNA repair. Furthermore, we show by immuno-precipitation assays that Rad50, Mre11 and the HerA helicase interact altogether.
Conclusion
Our analyses strongly support that in Sulfolobus acidocaldarius, the Mre11 protein and the RadA recombinase might play an active role in the repair of DNA damage introduced by gamma rays and/or may act as DNA damage sensors. Moreover, our results demonstrate the functional interaction between Mre11, Rad50 and the HerA helicase and suggest that each protein play different roles when acting on its own or in association with its partners. This report provides the first in vivo evidence supporting the implication of the Mre11 protein in DNA repair processes in the Archaea and showing its interaction with both Rad50 and the HerA bipolar helicase. Further studies on the functional interactions between these proteins, the NurA nuclease and the RadA recombinase, will allow us to define their roles and mechanism of action.
doi:10.1186/1471-2199-9-25
PMCID: PMC2288612  PMID: 18294364
3.  Structural and functional insights into DNA-end processing by the archaeal HerA helicase–NurA nuclease complex 
Nucleic Acids Research  2011;40(7):3183-3196.
Helicase–nuclease systems dedicated to DNA end resection in preparation for homologous recombination (HR) are present in all kingdoms of life. In thermophilic archaea, the HerA helicase and NurA nuclease cooperate with the highly conserved Mre11 and Rad50 proteins during HR-dependent DNA repair. Here we show that HerA and NurA must interact in a complex with specific subunit stoichiometry to process DNA ends efficiently. We determine crystallographically that NurA folds in a toroidal dimer of intertwined RNaseH-like domains. The central channel of the NurA dimer is too narrow for double-stranded DNA but appears well suited to accommodate one or two strands of an unwound duplex. We map a critical interface of the complex to an exposed hydrophobic epitope of NurA abutting the active site. Based upon the presented evidence, we propose alternative mechanisms of DNA end processing by the HerA-NurA complex.
doi:10.1093/nar/gkr1157
PMCID: PMC3326311  PMID: 22135300
4.  The P. furiosus Mre11/Rad50 complex promotes 5’ strand resection at a DNA double-strand break 
Cell  2008;135(2):250-260.
Summary
The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3’ single-stranded DNA for recombinase loading and strand exchange. In thermophilic archaea, the Mre11 and Rad50 genes cluster in an operon with genes encoding a helicase, HerA, and a 5’ to 3’ exonuclease, NurA, suggesting a common function. Here we show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5’ strand at a DNA end under physiological conditions in vitro. The 3’ single-stranded DNA generated by these enzymes can be utilized by the archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in archaea, and may serve as a model for DSB processing in eukaryotes.
doi:10.1016/j.cell.2008.09.054
PMCID: PMC2581932  PMID: 18957200
5.  The Structure of the NTPase That Powers DNA Packaging into Sulfolobus Turreted Icosahedral Virus 2 
Journal of Virology  2013;87(15):8388-8398.
Biochemical reactions powered by ATP hydrolysis are fundamental for the movement of molecules and cellular structures. One such reaction is the encapsidation of the double-stranded DNA (dsDNA) genome of an icosahedrally symmetric virus into a preformed procapsid with the help of a genome-translocating NTPase. Such NTPases have been characterized in detail from both RNA and tailed DNA viruses. We present four crystal structures and the biochemical activity of a thermophilic NTPase, B204, from the nontailed, membrane-containing, hyperthermoacidophilic archaeal dsDNA virus Sulfolobus turreted icosahedral virus 2. These are the first structures of a genome-packaging NTPase from a nontailed, dsDNA virus with an archaeal host. The four structures highlight the catalytic cycle of B204, pinpointing the molecular movement between substrate-bound (open) and empty (closed) active sites. The protein is shown to bind both single-stranded and double-stranded nucleic acids and to have an optimum activity at 80°C and pH 4.5. The overall fold of B204 places it in the FtsK-HerA superfamily of P-loop ATPases, whose cellular and viral members have been suggested to share a DNA-translocating mechanism.
doi:10.1128/JVI.00831-13
PMCID: PMC3719838  PMID: 23698307
6.  Crystal structure of the NurA–dAMP–Mn2+ complex 
Nucleic Acids Research  2011;40(5):2258-2270.
Generation of the 3′ overhang is a critical event during homologous recombination (HR) repair of DNA double strand breaks. A 5′–3′ nuclease, NurA, plays an important role in generating 3′ single-stranded DNA during archaeal HR, together with Mre11–Rad50 and HerA. We have determined the crystal structures of apo- and dAMP-Mn2+-bound NurA from Pyrococcus furiousus (Pf NurA) to provide the basis for its cleavage mechanism. Pf NurA forms a pyramid-shaped dimer containing a large central channel on one side, which becomes narrower towards the peak of the pyramid. The structure contains a PIWI domain with high similarity to argonaute, endoV nuclease and RNase H. The two active sites, each of which contains Mn2+ ion(s) and dAMP, are at the corners of the elliptical channel near the flat face of the dimer. The 3′ OH group of the ribose ring is directed toward the channel entrance, explaining the 5′–3′ nuclease activity of Pf NurA. We provide a DNA binding and cleavage model for Pf NurA.
doi:10.1093/nar/gkr999
PMCID: PMC3300031  PMID: 22064858
7.  Diversity of Virophages in Metagenomic Data Sets 
Journal of Virology  2013;87(8):4225-4236.
Virophages, e.g., Sputnik, Mavirus, and Organic Lake virophage (OLV), are unusual parasites of giant double-stranded DNA (dsDNA) viruses, yet little is known about their diversity. Here, we describe the global distribution, abundance, and genetic diversity of virophages based on analyzing and mapping comprehensive metagenomic databases. The results reveal a distinct abundance and worldwide distribution of virophages, involving almost all geographical zones and a variety of unique environments. These environments ranged from deep ocean to inland, iced to hydrothermal lakes, and human gut- to animal-associated habitats. Four complete virophage genomic sequences (Yellowstone Lake virophages [YSLVs]) were obtained, as was one nearly complete sequence (Ace Lake Mavirus [ALM]). The genomes obtained were 27,849 bp long with 26 predicted open reading frames (ORFs) (YSLV1), 23,184 bp with 21 ORFs (YSLV2), 27,050 bp with 23 ORFs (YSLV3), 28,306 bp with 34 ORFs (YSLV4), and 17,767 bp with 22 ORFs (ALM). The homologous counterparts of five genes, including putative FtsK-HerA family DNA packaging ATPase and genes encoding DNA helicase/primase, cysteine protease, major capsid protein (MCP), and minor capsid protein (mCP), were present in all virophages studied thus far. They also shared a conserved gene cluster comprising the two core genes of MCP and mCP. Comparative genomic and phylogenetic analyses showed that YSLVs, having a closer relationship to each other than to the other virophages, were more closely related to OLV than to Sputnik but distantly related to Mavirus and ALM. These findings indicate that virophages appear to be widespread and genetically diverse, with at least 3 major lineages.
doi:10.1128/JVI.03398-12
PMCID: PMC3624350  PMID: 23408616
8.  Genome Segregation and Packaging Machinery in Acanthamoeba polyphaga Mimivirus Is Reminiscent of Bacterial Apparatus 
Journal of Virology  2014;88(11):6069-6075.
ABSTRACT
Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms.
IMPORTANCE Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside the newly synthesized viral particle. These findings have important evolutionary implications about the origin and evolution of large viruses.
doi:10.1128/JVI.03199-13
PMCID: PMC4093880  PMID: 24623441
9.  Nanobiomotors of archaeal DNA repair machineries: current research status and application potential 
Cell & Bioscience  2014;4:32.
Nanobiomotors perform various important functions in the cell, and they also emerge as potential vehicle for drug delivery. These proteins employ conserved ATPase domains to convert chemical energy to mechanical work and motion. Several archaeal nucleic acid nanobiomotors, such as DNA helicases that unwind double-stranded DNA molecules during DNA damage repair, have been characterized in details. XPB, XPD and Hjm are SF2 family helicases, each of which employs two ATPase domains for ATP binding and hydrolysis to drive DNA unwinding. They also carry additional specific domains for substrate binding and regulation. Another helicase, HerA, forms a hexameric ring that may act as a DNA-pumping enzyme at the end processing of double-stranded DNA breaks. Common for all these nanobiomotors is that they contain ATPase domain that adopts RecA fold structure. This structure is characteristic for RecA/RadA family proteins and has been studied in great details. Here we review the structural analyses of these archaeal nucleic acid biomotors and the molecular mechanisms of how ATP binding and hydrolysis promote the conformation change that drives mechanical motion. The application potential of archaeal nanobiomotors in drug delivery has been discussed.
doi:10.1186/2045-3701-4-32
PMCID: PMC4080772  PMID: 24995126
Archaea; Nanobiomotor; Helicase; RecA fold; DNA repair
10.  The N-Terminal Membrane-Spanning Domain of the Escherichia coli DNA Translocase FtsK Hexamerizes at Midcell 
mBio  2013;4(6):e00800-13.
ABSTRACT
Bacterial FtsK plays a key role in coordinating cell division with the late stages of chromosome segregation. The N-terminal membrane-spanning domain of FtsK is required for cell division, whereas the C-terminal domain is a fast double-stranded DNA (dsDNA) translocase that brings the replication termination region of the chromosome to midcell, where it facilitates chromosome unlinking by activating XerCD-dif site-specific recombination. Therefore, FtsK coordinates the late stages of chromosome segregation with cell division. Although the translocase is known to act as a hexamer on DNA, it is unknown when and how hexamers form, as is the number of FtsK molecules in the cell and within the divisome. Using single-molecule live-cell imaging, we show that newborn Escherichia coli cells growing in minimal medium contain ~40 membrane-bound FtsK molecules that are largely monomeric; the numbers increase proportionately with cell growth. After recruitment to the midcell, FtsK is present only as hexamers. Hexamers are observed in all cells and form before any visible sign of cell constriction. An average of 7 FtsK hexamers per cell are present at midcell, with the N-terminal domain being able to hexamerize independently of the translocase. Detergent-solubilized and purified FtsK N-terminal domains readily form hexamers, as determined by in vitro biochemistry, thereby supporting the in vivo data. The hexameric state of the FtsK N-terminal domain at the division site may facilitate assembly of a functional C-terminal DNA translocase on chromosomal DNA.
IMPORTANCE
In the rod-shaped bacterium Escherichia coli, more than a dozen proteins act at the cell center to mediate cell division, which initiates while chromosome replication and segregation are under way. The protein FtsK coordinates cell division with the late stages of chromosome segregation. The N-terminal part of FtsK is membrane embedded and acts in division, while the C-terminal part forms a hexameric ring on chromosomal DNA, which the DNA can translocate rapidly to finalize chromosome segregation. Using quantitative live-cell imaging, which measures the position and number of FtsK molecules, we show that in all cells, FtsK hexamers form only at the cell center at the initiation of cell division. Furthermore, the FtsK N-terminal portion forms hexamers independently of the C-terminal translocase.
doi:10.1128/mBio.00800-13
PMCID: PMC3870252  PMID: 24302254
11.  Polar Positioning of a Conjugation Protein from the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis▿  
Journal of Bacteriology  2009;192(1):38-45.
ICEBs1 is an integrative and conjugative element found in the chromosome of Bacillus subtilis. ICEBs1 encodes functions needed for its excision and transfer to recipient cells. We found that the ICEBs1 gene conE (formerly yddE) is required for conjugation and that conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE. ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4. We found that a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells. At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles, as ConE-GFP was dispersed throughout the cytoplasm in a strain lacking ICEBs1. We also visualized the subcellular location of ICEBs1. When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell, a position characteristic of that chromosomal region. Following excision, ICEBs1 was more frequently found near a cell pole. Excision of ICEBs1 also caused altered positioning of at least one component of the replisome. Taken together, our findings indicate that ConE is a critical component of the ICEBs1 conjugation machinery, that conjugative transfer of ICEBs1 from B. subtilis likely initiates at a donor cell pole, and that ICEBs1 affects the subcellular position of the replisome.
doi:10.1128/JB.00860-09
PMCID: PMC2798270  PMID: 19734305
12.  FtsK translocation on DNA stops at XerCD-dif 
Nucleic Acids Research  2009;38(1):72-81.
Escherichia coli FtsK is a powerful, fast, double-stranded DNA translocase, which can strip proteins from DNA. FtsK acts in the late stages of chromosome segregation by facilitating sister chromosome unlinking at the division septum. KOPS-guided DNA translocation directs FtsK towards dif, located within the replication terminus region, ter, where FtsK activates XerCD site-specific recombination. Here we show that FtsK translocation stops specifically at XerCD-dif, thereby preventing removal of XerCD from dif and allowing activation of chromosome unlinking by recombination. Stoppage of translocation at XerCD-dif is accompanied by a reduction in FtsK ATPase and is not associated with FtsK dissociation from DNA. Specific stoppage at recombinase-DNA complexes does not require the FtsKγ regulatory subdomain, which interacts with XerD, and is not dependent on either recombinase-mediated DNA cleavage activity, or the formation of synaptic complexes.
doi:10.1093/nar/gkp843
PMCID: PMC2800217  PMID: 19854947
13.  Evidence for a Xer/dif System for Chromosome Resolution in Archaea 
PLoS Genetics  2010;6(10):e1001166.
Homologous recombination events between circular chromosomes, occurring during or after replication, can generate dimers that need to be converted to monomers prior to their segregation at cell division. In Escherichia coli, chromosome dimers are converted to monomers by two paralogous site-specific tyrosine recombinases of the Xer family (XerC/D). The Xer recombinases act at a specific dif site located in the replication termination region, assisted by the cell division protein FtsK. This chromosome resolution system has been predicted in most Bacteria and further characterized for some species. Archaea have circular chromosomes and an active homologous recombination system and should therefore resolve chromosome dimers. Most archaea harbour a single homologue of bacterial XerC/D proteins (XerA), but not of FtsK. Therefore, the role of XerA in chromosome resolution was unclear. Here, we have identified dif-like sites in archaeal genomes by using a combination of modeling and comparative genomics approaches. These sites are systematically located in replication termination regions. We validated our in silico prediction by showing that the XerA protein of Pyrococcus abyssi specifically recombines plasmids containing the predicted dif site in vitro. In contrast to the bacterial system, XerA can recombine dif sites in the absence of protein partners. Whereas Archaea and Bacteria use a completely different set of proteins for chromosome replication, our data strongly suggest that XerA is most likely used for chromosome resolution in Archaea.
Author Summary
Bacteria with circular chromosome and active homologous recombination systems have to resolve chromosomal dimers before segregation at cell division. In Escherichia coli, the Xer site-specific recombination system, composed of two recombinases and a specific chromosomal site (dif), is involved in the correct inheritance of the chromosome. The recombination event is tightly regulated by the chromosome translocase FtsK. This chromosome resolution system has been predicted in most bacteria and further characterized for some species. Intriguingly, most archaea possess a gene coding for a recombinase homologous to bacterial Xers, but none have homologues of the bacterial FtsK. We identified the specific target sites for archaeal Xer. This site, present in one copy per chromosome, is located in the replication termination region and shows sequence similarities with bacterial dif sites. In vitro, the archaeal Xer recombines this site in the absence of protein partner. It has been shown that DNA–related proteins from Archaea and Eukarya share a common origin, whereas their analogues in Bacteria have evolved independently. In this context, Eukarya and Archaea would represent sister groups. Therefore, the presence of a shared Xer-dif system between Bacteria and Archaea illustrates the complex origin of modern DNA genomes.
doi:10.1371/journal.pgen.1001166
PMCID: PMC2958812  PMID: 20975945
14.  Comparative genomic and transcriptional analyses of CRISPR systems across the genus Pyrobaculum 
Within the domain Archaea, the CRISPR immune system appears to be nearly ubiquitous based on computational genome analyses. Initial studies in bacteria demonstrated that the CRISPR system targets invading plasmid and viral DNA. Recent experiments in the model archaeon Pyrococcus furiosus have uncovered a novel RNA-targeting variant of the CRISPR system. Because our understanding of CRISPR system evolution in other archaea is limited, we have taken a comparative genomic and transcriptomic view of the CRISPR arrays across six diverse species within the crenarchaeal genus Pyrobaculum. We present transcriptional data from each of four species in the genus (P. aerophilum, P. islandicum, P. calidifontis, P. arsenaticum), analyzing mature CRISPR-associated small RNA abundance from over 20 arrays. Within the genus, there is remarkable conservation of CRISPR array structure, as well as unique features that are have not been studied in other archaeal systems. These unique features include: a nearly invariant CRISPR promoter, conservation of direct repeat families, the 5′ polarity of CRISPR-associated small RNA abundance, and a novel CRISPR-specific association with homologues of nurA and herA. These analyses provide a genus-level evolutionary perspective on archaeal CRISPR systems, broadening our understanding beyond existing non-comparative model systems.
doi:10.3389/fmicb.2012.00251
PMCID: PMC3396285  PMID: 22811677
Pyrobaculum; CRISPR; sRNA; crRNA; repeat; RNAseq
15.  Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera 
Nucleic Acids Research  2013;41(12):6259-6272.
DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.
doi:10.1093/nar/gkt323
PMCID: PMC3695512  PMID: 23625962
16.  Viral and cellular SOS-regulated motor proteins: dsDNA translocation mechanisms with divergent functions 
Cell & Bioscience  2014;4:31.
DNA damage attacks on bacterial cells have been known to activate the SOS response, a transcriptional response affecting chromosome replication, DNA recombination and repair, cell division and prophage induction. All these functions require double-stranded (ds) DNA translocation by ASCE hexameric motors. This review seeks to delineate the structural and functional characteristics of the SOS response and the SOS-regulated DNA translocases FtsK and RuvB with the phi29 bacteriophage packaging motor gp16 ATPase as a prototype to study bacterial motors. While gp16 ATPase, cellular FtsK and RuvB are similarly comprised of hexameric rings encircling dsDNA and functioning as ATP-driven DNA translocases, they utilize different mechanisms to accomplish separate functions, suggesting a convergent evolution of these motors. The gp16 ATPase and FtsK use a novel revolution mechanism, generating a power stroke between subunits through an entropy-DNA affinity switch and pushing dsDNA inward without rotation of DNA and the motor, whereas RuvB seems to employ a rotation mechanism that remains to be further characterized. While FtsK and RuvB perform essential tasks during the SOS response, their roles may be far more significant as SOS response is involved in antibiotic-inducible bacterial vesiculation and biofilm formation as well as the perspective of the bacteria-cancer evolutionary interaction.
doi:10.1186/2045-3701-4-31
PMCID: PMC4080785  PMID: 24995125
AAA+ proteins; DNA translocases; DNA repair; Replication; Recombination; SOS response; Bacteria; Phages
17.  Membrane-associated DNA Transport Machines 
DNA pumps play important roles in bacteria during cell division and during the transfer of genetic material by conjugation and transformation. The FtsK/SpoIIIE proteins carry out the translocation of double-stranded DNA to ensure complete chromosome segregation during cell division. In contrast, the complex molecular machines that mediate conjugation and genetic transformation drive the transport of single stranded DNA. The transformation machine also processes this internalized DNA and mediates its recombination with the resident chromosome during and after uptake, whereas the conjugation apparatus processes DNA before transfer. This article reviews these three types of DNA pumps, with attention to what is understood of their molecular mechanisms, their energetics and their cellular localizations.
The FtsK/SpoIIIE DNA pumps help segregate chromosomes during bacterial cell division. Other pumps, such as DTR, transport single-stranded DNA into cells during conjugation and transformation.
doi:10.1101/cshperspect.a000406
PMCID: PMC2890206  PMID: 20573715
18.  Genetic Interactions of smc, ftsK, and parB Genes in Streptomyces coelicolor and Their Developmental Genome Segregation Phenotypes▿ † 
Journal of Bacteriology  2008;191(1):320-332.
The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for Streptomyces coelicolor, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for ftsK, smc, and parB. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (structural maintenance of chromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an ftsK-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the smc and ftsK deletion-insertion mutants (ftsK′ truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described parB-null mutant (12% anucleate spores). parB and smc were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the smc parB mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that scpA and scpAB mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid.
doi:10.1128/JB.00858-08
PMCID: PMC2612423  PMID: 18978061
19.  The translational landscape of the splicing factor SRSF1 and its role in mitosis 
eLife  2014;3:e02028.
The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer.
DOI: http://dx.doi.org/10.7554/eLife.02028.001
eLife digest
Genes contain the instructions to make proteins. These instructions are first transcribed to produce an intermediate molecule called a messenger RNA (mRNA), which is then translated to produce the protein. However, gene sequences are often interrupted by ‘introns’, sections of DNA that do not code for protein, and these introns must be removed from the mRNA molecules via a process called ‘splicing’ before the protein is produced.
Splicing can also be used to ‘mix and match’ sections of gene sequences to produce slightly different versions of the same protein in a process called ‘alternative splicing’. SRSF1 is one of a family of proteins that control both types of gene splicing but also promotes the translation of specific mRNAs. To date only a few of the genes whose translation is regulated by SRSF1 have been identified.
Here, Maslon, Heras et al. have used human cells that artificially produce more SRSF1 protein than normal to identify those genes whose translation is regulated by SRSF1. Over 1500 ‘target genes’ were found; many of which encoded proteins that are involved in cell division—and cells with less SRSF1 than normal failed to divide properly. Maslon, Heras et al. also found a link between alternative splicing and protein translation: many of the mRNAs that were spliced differently in cells that over-produced SRSF1 were also genes whose translation was affected by SRSF1.
Since uncontrolled cell division, or defects in mRNA splicing or protein synthesis are all often linked to cancer, these discoveries might provide new insights into the mechanisms underlying this disease.
DOI: http://dx.doi.org/10.7554/eLife.02028.002
doi:10.7554/eLife.02028
PMCID: PMC4027812  PMID: 24842991
translation; splicing; SR proteins; human
20.  Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair 
Nucleic Acids Research  2014;42(8):4996-5006.
Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.
doi:10.1093/nar/gku160
PMCID: PMC4005673  PMID: 24589584
21.  Localization of Cell Division Protein FtsK to the Escherichia coli Septum and Identification of a Potential N-Terminal Targeting Domain 
Journal of Bacteriology  1998;180(5):1296-1304.
Escherichia coli cell division protein FtsK is a homolog of Bacillus subtilis SpoIIIE and appears to act late in the septation process. To determine whether FtsK localizes to the septum, we fused three N-terminal segments of FtsK to green fluorescent protein (GFP) and expressed them in E. coli cells. All three segments were sufficient to target GFP to the septum, suggesting that as little as the first 15% of the protein is a septum-targeting domain. Localized fluorescence was detectable only in cells containing a visible midcell constriction, suggesting that FtsK targeting normally occurs only at a late stage of septation. The largest two FtsK-GFP fusions were able at least partially to complement the ftsK44 mutation in trans, suggesting that the N- and C-terminal domains are functionally separable. However, overproduction of FtsK-GFP resulted in a late-septation phenotype similar to that of ftsK44, with fluorescent dots localized at the blocked septa, suggesting that high levels of the N-terminal domain may still localize but also inhibit FtsK activity. Interestingly, under these conditions fluorescence was also sometimes localized as bands at potential division sites, suggesting that FtsK-GFP is capable of targeting very early. In addition, FtsK-GFP localized to potential division sites in cephalexin-induced and ftsI mutant filaments, further supporting the idea that FtsK-GFP can target early, perhaps by recognizing FtsZ directly. This hypothesis was supported by the failure of FtsK-GFP to localize in ftsZ mutant filaments. In ftsK44 mutant filaments, FtsA and FtsZ were usually localized to potential division sites between the blocked septa. When the ftsK44 mutation was incorporated into the FtsK-GFP fusions, localization to midcell ranged between very weak and undetectable, suggesting that the FtsK44 mutant protein is defective in targeting the septum.
PMCID: PMC107020  PMID: 9495771
22.  Structure and DNA-binding properties of the Bacillus subtilis SpoIIIE DNA translocase revealed by single-molecule and electron microscopies 
Nucleic Acids Research  2013;42(4):2624-2636.
SpoIIIE/FtsK are a family of ring-shaped, membrane-anchored, ATP-fuelled motors required to segregate DNA across bacterial membranes. This process is directional and requires that SpoIIIE/FtsK recognize highly skewed octameric sequences (SRS/KOPS for SpoIIIE/FtsK) distributed along the chromosome. Two models have been proposed to explain the mechanism by which SpoIIIE/FtsK interact with DNA. The loading model proposes that SpoIIIE/FtsK oligomerize exclusively on SpoIIIE recognition sequence/orienting polar sequences (SRS/KOPS) to accomplish directional DNA translocation, whereas the target search and activation mechanism proposes that pre-assembled SpoIIIE/FtsK hexamers bind to non-specific DNA, reach SRS/KOPS by diffusion/3d hopping and activate at SRS/KOPS. Here, we employ single-molecule total internal reflection imaging, atomic force and electron microscopies and ensemble biochemical methods to test these predictions and obtain further insight into the SpoIIIE–DNA mechanism of interaction. First, we find that SpoIIIE binds DNA as a homo-hexamer with neither ATP binding nor hydrolysis affecting the binding mechanism or affinity. Second, we show that hexameric SpoIIIE directly binds to double-stranded DNA without requiring the presence of SRS or free DNA ends. Finally, we find that SpoIIIE hexamers can show open and closed conformations in solution, with open-ring conformations most likely resembling a state poised to load to non-specific, double-stranded DNA. These results suggest how SpoIIIE and related ring-shaped motors may be split open to bind topologically closed DNA.
doi:10.1093/nar/gkt1231
PMCID: PMC3936747  PMID: 24297254
23.  Asymmetric DNA requirements in Xer recombination activation by FtsK 
Nucleic Acids Research  2009;37(7):2371-2380.
In bacteria with circular chromosomes, homologous recombination events can lead to the formation of chromosome dimers. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover by two tyrosine recombinases, XerC and XerD, at a specific site on the chromosome, dif. Recombination depends on a direct contact between XerD and a cell division protein, FtsK, which functions as a hexameric double stranded DNA translocase. Here, we have investigated how the structure and composition of DNA interferes with Xer recombination activation by FtsK. XerC and XerD each cleave a specific strand on dif, the top and bottom strand, respectively. We found that the integrity and nature of eight bottom-strand nucleotides and three top-strand nucleotides immediately adjacent to the XerD-binding site of dif are crucial for recombination. These nucleotides are probably not implicated in FtsK translocation since FtsK could translocate on single stranded DNA in both the 5′–3′ and 3′–5′ orientation along a few nucleotides. We propose that they are required to stabilize FtsK in the vicinity of dif for recombination to occur because the FtsK–XerD interaction is too transient or too weak in itself to allow for XerD catalysis.
doi:10.1093/nar/gkp104
PMCID: PMC2673442  PMID: 19246541
24.  FtsK-Dependent Dimer Resolution on Multiple Chromosomes in the Pathogen Vibrio cholerae 
PLoS Genetics  2008;4(9):e1000201.
Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.
Author Summary
During proliferation, DNA synthesis, chromosome segregation, and cell division must be coordinated to ensure the stable inheritance of the genetic material. In eukaryotes, this is achieved by checkpoint mechanisms that delay certain steps until others are completed. No such temporal separation exists in bacteria, which can undergo overlapping replication cycles. The eukaryotic cell cycle is particularly well suited to the management of multiple chromosomes, with the same replication initiation and segregation machineries operating on all the chromosomes, while the bacterial cell cycle is linked to genomes of less complexity, most bacteria harboring a single chromosome. The discovery of bacteria harboring multiple circular chromosomes, such as V. cholerae, raised therefore a considerable interest for the mechanisms ensuring the synchronous management of different replicons. Here, we took advantage of our knowledge of chromosome dimer resolution, the only bacterial segregation process for which coordination with cell division is well understood, to investigate one of the mechanisms ensuring the synchronous management of the smaller, plasmid-like, and larger, chromosome-like, replicons of V. cholerae.
doi:10.1371/journal.pgen.1000201
PMCID: PMC2533119  PMID: 18818731
25.  Sequencing and Characterization of pBM400 from Bacillus megaterium QM B1551 
Applied and Environmental Microbiology  2003;69(11):6888-6898.
Bacillus megaterium QM B1551 plasmid pBM400, one of seven indigenous plasmids, has been labeled with a selectable marker, isolated, completely sequenced, and partially characterized. A sequence of 53,903 bp was generated, revealing a total of 50 predicted open reading frames (ORFs); 33 were carried on one strand and 17 were carried on the other. These ORFs comprised 57% of the pBM400 sequence. Besides the replicon region and a complete rRNA operon that have previously been described, several interesting genes were found, including genes for predicted proteins for cell division (FtsZ and FtsK), DNA-RNA interaction (FtsK, Int/Rec, and reverse transcriptase), germination (CwlJ), styrene degradation (StyA), and heavy metal resistance (Cu-Cd export and ATPase). Three of the ORF products had high similarities to proteins from the Bacillus anthracis virulence plasmid pXO1. An insertion element with similarity to the IS256 family and several hypothetical proteins similar to those from the chromosomes of other Bacillus and Lactococcus species were present. This study provides a basis for isolation and sequencing of other high-molecular-weight plasmids from QM B1551 and for understanding the role of megaplasmids in gram-positive bacteria. The genes carried by pBM400 suggest a possible role of this plasmid in the survival of B. megaterium in hostile environments with heavy metals or styrene and also suggest that there has been an exchange of genes within the gram-positive bacteria, including pathogens.
doi:10.1128/AEM.69.11.6888-6898.2003
PMCID: PMC262321  PMID: 14602653

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