Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK - PubMed (original) (raw)

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

Ja Yil Lee et al. Proc Natl Acad Sci U S A. 2012.

Abstract

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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Schematic representation of (A) nanofabricated double-tethered DNA curtain assay, (B) λ phage DNA bearing the two native 1xKOPS sites and the engineered 3xKOPS site, and (C) the covalently linked FtsK trimer with an N-terminal biotin tag. (D) Images of a double-tethered YOYO1-stained DNA curtain (green) bound by QD-tagged FtsK (magenta). The DNA, FtsK, and corresponding overlay are shown at Top, Middle, and Bottom, respectively.

Fig. 2.

(A) Kymogram illustrating ATP-dependent translocation of QD-tagged FtsK (magenta) along a single DNA molecule (unlabeled). Transient gaps in the magenta signal correspond to QD blinking. The location of the KOPS sites is illustrated schematically on the left, and arrowheads indicate the arbitrarily assigned (+) and (-) designations for translocation direction. (B) Example of FtsK bound to DNA in the presence of ATPγS. (C) Velocity distribution with data segregated into (+) and (−) directions. (D) Scatter plot showing the relationship between (+) and (−) direction velocities for individual molecules of translocating FtsK. The red line illustrates a fit to the data and yields a slope of 1. (E) Velocity distribution histogram comprising the combined (+) and (−) velocity data sets, revealing a mean velocity of V = 4.66 ± 1.3 kb s−1. (F) Translocation velocities at varying concentrations of ATP. (G) Velocity distributions for data collected at 37 °C and 5 mM ATP, revealing a mean velocity of V = 17.5 ± 3.5 kb s−1.

Fig. 3.

(A–F) Binding distribution histograms for QD-tagged FtsK in the presence of the indicated nucleotide cofactor. All reactions were conducted in buffer containing 100 mM NaCl. For D and F, the reported distribution corresponds to the first detected binding position of FtsK molecules before their translocation away from the initial binding sites (

Fig. S9

and

SI Materials and Methods

). The relative locations of the KOPS sites are illustrated schematically in A.

Fig. 4.

KOPS recognition by ATPase defective mutants of FtsK. (A) Kymograms (Upper) for the FtsK 3xK997A Walker A mutant and the Walker B 3xD1121A mutant. (B) Binding distributions for the K997A FtsK Walker A mutants, as indicated. (C) Binding distributions of D1121A mutants, as indicated. All reactions used 10 pM mutated FtsK trimer and contained 100 mM NaCl. The relative number and location of the mutant and WT subunits within the FtsK hexamers are schematically illustrated in B and C.

Fig. 5.

Real-time visualization of KOPS targeting by FtsK. (A) Kymogram showing the initial association of FtsK with the λ-phage substrate in a reaction containing 1 mM ADP. Examples of nonspecific and KOPS-specific binding are highlighted and shown along with the corresponding particle tracking data. (B) Binding distribution lifetimes of FtsK bound to KOPS. (C) Binding distribution lifetimes of FtsK bound to nonspecific sites. Specific and nonspecific binding data were collected at 1 mM ADP, and black lines correspond to single exponential fits to the data.

Fig. 6.

KOPS dictates translocation direction only upon initial loading. (A) Examples of tracking data showing that FtsK leaves KOPS in the direction dictated by the orientation of KOPS. The relative orientation of each is depicted with arrows, and the location of each KOPS site is indicated with a dashed orange line. Fifteen seconds of each trajectory are shown. (B and C) Histograms showing (B) positions at which FtsK changed direction or (C) paused. Pauses were defined as five consecutive frames with no detectable movement. The first and last bins were excluded from the binding distributions in B and C to avoid biasing the data due to FtsK collisions with the barriers.

References

1. Sherratt DJ, Arciszewska LK, Crozat E, Graham JE, Grainge I. The Escherichia coli DNA translocase FtsK. Biochem Soc Trans. 2010;38:395–398. - PubMed
1. Barre FX. FtsK and SpoIIIE: The tale of the conserved tails. Mol Microbiol. 2007;66:1051–1055. - PubMed
1. Carnoy C, Roten CA. The dif/Xer recombination systems in proteobacteria. PLoS ONE. 2009;4:e6531. - PMC - PubMed
1. Kono N, Arakawa K, Tomita M. Comprehensive prediction of chromosome dimer resolution sites in bacterial genomes. BMC Genomics. 2011;12:19. - PMC - PubMed
1. Massey TH, Mercogliano CP, Yates J, Sherratt DJ, Löwe J. Double-stranded DNA translocation: Structure and mechanism of hexameric FtsK. Mol Cell. 2006;23:457–469. - PubMed

Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK - PubMed (original) (raw)