Asymmetric activation of Xer site-specific recombination by FtsK

Abstract

Chromosome dimers, which frequently form in Escherichia coli, are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific site on the chromosome, dif, together with the cell division protein FtsK. The C-terminal domain of FtsK (FtsK(C)) is a DNA translocase implicated in helping synapsis of the dif sites and in locally promoting XerD strand exchanges after synapse formation. Here we show that FtsK(C) ATPase activity is directly involved in the local activation of Xer recombination at dif, by using an intermolecular recombination assay that prevents significant DNA translocation, and we confirm that FtsK acts before Holliday junction formation. We show that activation only occurs with a DNA segment adjacent to the XerD-binding site of dif. Only one such DNA extension is required. Taken together, our data suggest that FtsK needs to contact the XerD recombinase to switch its activity on using ATP hydrolysis.

Figure 1

Minimal XerCD–FtsK recombination system. (A) Schematic illustrating an intermolecular Xer recombination reaction between a 411 bp unlabelled DNA fragment containing a centrally located dif and a short, dif-containing DNA (60 bp) 5′-end-labelled with ³²P on both strands. (B) Intermolecular recombination by XerCD is absolutely dependent on FtsK_50C and ATP. Reactions were incubated at 37°C for 60 min and analysed by 7% TBE–PAGE.

Figure 2

FtsK-dependent Xer recombination requires DNA adjacent to the XerD-binding site only. The schematic illustrates reactions between short, labelled dif-containing DNAs and unlabelled DNAs with either (1) two arms flanking dif or one arm adjacent to dif on either the XerD side (2) or the XerC side (3). Recombination reactions were incubated at 37°C for 60 min and analysed by 7% TBE–PAGE.

Figure 3

Asymmetric DNA requirement of FtsK_50C is independent of DNA sequence. (A) Orientation of dif sites in pFX315 and pFX316, and the recombination products expected from reactions between dif-containing restriction fragments and a short, labelled dif-containing DNA (3CD3). Lowercase letters indicate DNA orientation. The single RAG sequence element is annotated. (B) Electrophoretic analysis of 60 min reactions detailed in (A). (C) Recombination over time for reactions between short, dif-containing DNAs where either one or both short fragments have a Dside extension. The reactions with just one D-side arm are between 3CD3 and 315 M/N.

Figure 4

Effects of DNA arm length and backbone continuity on FtsK_50C activities. (A) Unlabelled DNAs with different lengths of arm adjoining either the XerC side or the XerD side of dif were reacted with short, labelled dif-containing DNAs with either 3-bp arms (left panel) or 3 bp on the XerC side and 16 bp on the XerD side (3CD16; right panel), in the presence of XerCD, FtsK_50C and ATP for 60 min. (B) Histogram comparing the levels of recombination observed when unlabelled DNAs with XerC side (white bars) or XerD side (grey bars) extensions were reacted with labelled 3CD16. The results are the means of three experiments. (C) Graph of DNA length against FtsK_50C ATPase activity (mean turnover±s.d. from three independent reactions, in the absence of XerCD). (D) A singlestrand nick does not affect FtsK_50C activation of recombination. Unlabelled DNA without (top reaction) or with (bottom reaction) a single-stranded nick 3 bp along the XerD DNA arm was reacted with labelled 3CD3 in the presence of XerCD, FtsK_50C and ATP for 60 min. The results are the mean±s.d. of three independent experiments.

Figure 5

FtsK_50C does not affect the direction or efficiency of resolution of a dif-containing HJ. The HJ schematized was prepared by annealing four oligonucleotides and then reacted with XerCD, in the presence or absence of FtsK_50C and ATP, for 60 min at 37°C. Reactions were deproteinized before 6% TBE–PAGE.