Identification of oligonucleotide sequences that direct the movement of the Escherichia coli FtsK translocase - PubMed (original) (raw)

Identification of oligonucleotide sequences that direct the movement of the Escherichia coli FtsK translocase

Oren Levy et al. Proc Natl Acad Sci U S A. 2005.

Abstract

FtsK from Escherichia coli is a fast and sequence-directed DNA translocase with roles in chromosome dimer resolution, segregation, and decatenation. From the movement of single FtsK particles on defined DNA substrates and an analysis of skewed DNA sequences in bacteria, we identify GNGNAGGG, its complement, or both as a sequence motif that controls translocation directionality. GNGNAGGG is skewed so that it is predominantly on the leading strand of chromosomal replication. Translocation across this octamer from the 3' side of the G-rich strand causes FtsK to pause, turn around, and translocate in the opposite direction. Only 39 +/- 4% of the encounters between FtsK and the octamer result in a turnaround, congruent with our optimum turnaround probability prediction of 30%. The probability that the observed skew of GNGNAGGG within 1 megabase of dif occurred by chance in E. coli is 1.7 x 10(-57), and similarly dramatic skews are found in the five other bacterial genomes we examined. The fact that FtsK acts only in the terminus region and the octamer skew extends from origin to terminus implies that this skew is also important in other basic cellular processes that are common among bacteria. Finally, we show that the FtsK translocase is a powerful motor that is able to displace a triplex-forming oligo from a DNA substrate.

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Figures

Fig. 1.

FtsK pauses and turns around at specific points in the dif region of the E. coli chromosome. (A) Histogram of turnaround locations shows that 88% (486/555) of turnarounds occur when FtsK translocates away from dif (n = 555 over three tethers). Red indicates movement from right to left; blue represents movement from left to right. Location and orientation of GNGNAGGG and RGNAGGGS motifs are indicated above the observed turnaround zones (I–IV). (B) Dwell time histogram of FtsK on the same tether as in A indicates that FtsK pauses at the turnaround zones. (C) Histogram of pause time (n = 193 on three tethers) observed at turnaround zones I or II has a mean of 1.0 ± 0.1 sec.

Fig. 2.

GNGNAGGG motif is the top FRS candidate. (A) The probability that the skew of GNGNAGGG occurred by chance (P value) at 15 kb, 100 kb, and 1 Mb away from dif. This probability is extremely small (P = 1.7 × 10–57) at 1 Mb away from dif.(B) Walk diagrams for GNGNAGGG motif display a clear skew bias centered on dif for all six genomes examined. An eight-base window is shifted across the genome, and the trace moves up by one when GNGNAGGG is encountered and down by one when its complement occurs.

Fig. 3.

FtsK50C recognizes the GGGCAGGGG (FRS) when approaching from the 3′ end of the G-rich strand and reverses direction. (A) Schematic representation of the tethers and optical tweezers experiment. Tethers are ≈48 kb long, consisting of 41 kb from lambda phage, a 6-kb plasmid DNA as a spacer, and the test sequence located between them. FtsK approaches from the bottom. In the anti orientation, FtsK (blue circle) approaches the test sequence (red arrow) from the 3′ end of the G-rich strand, and in the iso orientation, it approaches from the opposite side. (B) FtsK preferentially recognizes an FRS in the anti orientation. Test sequences consist of one or five FRSs in the iso orientation (iso 1-mer and iso 5-mer), one or five FRSs in the anti orientation (anti 1-mer and anti 5-mer), five scrambled FRSs (GGAGGCGGG) in the anti orientation (scrambled 5-mer), five RAG sequences (GGCAGGGG), and five GNGNAGGG sequences (GGGCAGGG). (C) Representative trace of a single FtsK translocation on anti 5-mer (Left) and anti 1-mer (Right). (D) Representative trace for three separate FtsK translocation events on iso 5-mer (Left) and iso 1-mer (Right). Location of test sequence is indicated with a gray dotted line.

Fig. 4.

Theoretical optimal turnaround probability for FtsK as it translocates over an FRS is congruent with measured values. The expected time to reach dif (Eq. 1) is plotted against the turnaround probability; assuming FtsK binds 100 kb away from dif, the FRS density is 1/3 kb–1, and the skew is 80, 85, 90, or 95. Squares show the minimum, and circles show the simplified optimal turnaround probability as defined by Eq. 2.

Fig. 5.

FtsK50C is a molecular wirestripper. (A) Schematic depicting FtsK50C displacing a DNA triplex substrate. FtsK50C (blue circle) likely binds within the 3-kb duplex region because of relative size, and translocation into the DNA triplex (jagged line) leads to triplex displacement. (B) Triplex displacement by FtsK requires ATP hydrolysis. Varying amounts (0–152 nM) of FtsK50C were incubated for 15 min at 25°C with 3 mM ATP (lanes 1–4), no ATP or 3 mM ATPγS with 152 nM FtsK50C controls (lanes 5 and 6), or no ATP and no FtsK control (lane 7). Lane 8 is identical to lane 4, but the reaction mixture was heat-denatured at 75°C for 5 min before loading. (C) Molecular wirestripping by FtsK50C is controlled by its directionality sequence. Schematic diagram of DNA triplex substrates indicating the location of directionality sequences (top). DNA triplex displacement reactions were performed as described in Materials and Methods, using substrates with 5-mer repeats of the sequence GGGCAGGGG in iso orientation (black), anti orientation (red), or the partially scrambled sequence GGAGGCGGG in the anti orientation (blue).

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Identification of oligonucleotide sequences that direct the movement of the Escherichia coli FtsK translocase - PubMed (original) (raw)