Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis

Ptacin, Jerod L.; Nollmann, Marcelo; Becker, Eric; Cozzarelli, Nicholas R.; Pogliano, Kit; Bustamante, Carlos

doi:10.1038/nsmb.1412

Cited by 92 publications

(176 citation statements)

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“…For these experiments we used the soluble motor domain of SpoIIIE, which has been shown to translocate along DNA in vitro (Bath et al 2000;Ptacin et al 2008). E. coli RNA polymerase containing 70 was assembled on a fluorescently labeled DNA substrate containing the PR promoter.…”

Section: The Atpase Domain Of Spoiiie Can Displace Rna Polymerase Boumentioning

confidence: 99%

“…Displacement of stalled RNA polymerase by SpoIIIE was assayed by incubating 10 nM stalled elongation complex with 420 nM of the SpoIIIE motor domain (SpoIIIE C ) (Ptacin et al 2008) and 3 mM ATP in SpoIIIE C reaction buffer (50 mM Tris-HCl at pH 7.5, 10 mM MgCl 2 ). All reactions were incubated for 10 min at 37°C and then stopped by incubation with 0.2 mg/mL heparin for 5 min at 22°C.…”

Section: Rna Polymerase Stalled Elongation Complex Displacement Assaysmentioning

confidence: 99%

“…These DNA transporters are polytopic membrane proteins with cytoplasmic ATPase domains. In single molecule experiments, the soluble ATPase domains (the motor domain) of these proteins can translocate along double-stranded DNA at a rate of ∼4-7 kbp/sec (Saleh et al 2004;Pease et al 2005;Ptacin et al 2008). The crystal structure of the cytoplasmic motor domain of FtsK indicates that it assembles into hexameric rings that are large enough to accommodate dsDNA (Massey et al 2006).…”

mentioning

confidence: 99%

“…The pore size of the FtsK hexameric ring in the crystal structure is ∼30 Å. Doublestranded DNA could easily be threaded through this annulus, but proteins associated with the DNA might not be accommodated. In vitro experiments with the cytoplasmic motor domains of FtsK and SpoIIIE indicate that these enzymes can displace ssDNA when it encounters a DNA triplex (Bigot et al 2005;Levy et al 2005;Ptacin et al 2008), suggesting that DNA-binding proteins might also be stripped off. However, some DNA-binding proteins have very high affinity for their cognate binding sites.…”

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confidence: 99%

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SpoIIIE strips proteins off the DNA during chromosome translocation

Marquis¹,

Nollmann²,

Ptacin³

et al. 2008

Genes Dev.

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The FtsK/SpoIIIE family of DNA transporters are responsible for translocating missegregated chromosomes after the completion of cell division. An extreme example of this post-cytokinetic DNA segregation occurs during spore formation in the bacterium Bacillus subtilis, where SpoIIIE pumps three-quarters of the chromosome (>3 megabases) into one of the two daughter cells. Here, we investigate the fate of the proteins associated with the translocated DNA. Taking advantage of several unique features of Bacillus sporulation, we demonstrate that RNA polymerase, transcription factors, and chromosome remodeling proteins are stripped off the DNA during translocation of the chromosome into the forespore compartment. Furthermore, we show that in vitro the soluble ATPase domain of SpoIIIE can displace RNA polymerase bound to DNA, suggesting that SpoIIIE alone is capable of this wire-stripping activity. Our data suggest that the bulk of the forespore chromosome is translocated naked into the forespore compartment. We propose that the translocation-stripping activity of SpoIIIE plays a key role in reprogramming developmental gene expression in the forespore.[Keywords: Chromosome segregation; chromosome translocation; sporulation; FtsK; AAA+ ATPase] Supplemental material is available at http://www.genesdev.org.

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Section: The Atpase Domain Of Spoiiie Can Displace Rna Polymerase Boumentioning

confidence: 99%

Section: Rna Polymerase Stalled Elongation Complex Displacement Assaysmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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SpoIIIE strips proteins off the DNA during chromosome translocation

Marquis¹,

Nollmann²,

Ptacin³

et al. 2008

Genes Dev.

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“…In vitro work has focused on the C-terminal FtsKαβγ motor domain, referred to as FtsK 50C , or similar constructs (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). FtsK 50C is a DNA-dependent ATPase, which can activate XerCDdif recombination (6).…”

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confidence: 99%

Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK

Lee¹,

Finkelstein²,

Crozat

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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FtsK is a hexameric DNA translocase that participates in the final stages of bacterial chromosome segregation. Here we investigate the DNA-binding and translocation activities of FtsK in real time by imaging fluorescently tagged proteins on nanofabricated curtains of DNA. We show that FtsK preferentially loads at 8-bp KOPS (FtsK Orienting Polar Sequences) sites and that loading is enhanced in the presence of ADP. We also demonstrate that FtsK locates KOPS through a mechanism that does not involve extensive 1D diffusion at the scale of our resolution. Upon addition of ATP, KOPS-bound FtsK translocates in the direction dictated by KOPS polarity, and once FtsK has begun translocating it does not rerecognize KOPS from either direction. However, FtsK can abruptly change directions while translocating along DNA independent of KOPS, suggesting that the ability to reorient on DNA does not arise from DNA sequence-specific effects. Taken together, our data support a model in which FtsK locates KOPS through random collisions, preferentially engages KOPS in the ADP-bound state, translocates in the direction dictated by the polar orientation of KOPS, and is incapable of recognizing KOPS while translocating along DNA.

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FtsK DNA Translocase: The Fast Motor That Knows Where It's Going

Crozat

Grainge

2010

ChemBioChem

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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.

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Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis

Cited by 92 publications

References 48 publications

SpoIIIE strips proteins off the DNA during chromosome translocation

SpoIIIE strips proteins off the DNA during chromosome translocation

Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK

FtsK DNA Translocase: The Fast Motor That Knows Where It's Going

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