Circles: the replication-recombination-chromosome segregation connection - PubMed (original) (raw)

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Circles: the replication-recombination-chromosome segregation connection

F X Barre et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Crossing over by homologous recombination between monomeric circular chromosomes generates dimeric circular chromosomes that cannot be segregated to daughter cells during cell division. In Escherichia coli, homologous recombination is biased so that most homologous recombination events generate noncrossover monomeric circular chromosomes. This bias is lost in ruv mutants. A novel protein, RarA, which is highly conserved in eubacteria and eukaryotes and is related to the RuvB and the DnaX proteins, gamma and tau, may influence the formation of crossover recombinants. Those dimeric chromosomes that do form are converted to monomers by Xer site-specific recombination at the recombination site dif, located in the replication terminus region of the E. coli chromosome. The septum-located FtsK protein, which coordinates cell division with chromosome segregation, is required for a complete Xer recombination reaction at dif. Only correctly positioned dif sites present in a chromosomal dimer are able to access septum-located FtsK. FtsK acts by facilitating a conformational change in the Xer recombination Holliday junction intermediate formed by XerC recombinase. This change provides a substrate for XerD, which then completes the recombination reaction.

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Figures

Figure 1

Figure 1

A scheme to illustrate stalled replication fork rebuilding by replication fork regression, by using recombination enzymes in the E. coli chromosome (adapted from refs. , , , and 25). Pathways that lead to a single crossover (or an odd number of crossovers) generate dimeric chromosomes, and those that act without crossing over (or even numbers of crossovers) retain the monomeric status of the chromosome. Pathways considered to be major routes to retaining the monomeric chromosome status are overlaid onto a beige background. Similarly, the arrows bounded by a bold line are intended to indicate major pathways, with relative contributions being indicated by arrow breadth. Those arrows in dark green indicate pathways that would be expected to be RecABCD-dependent, whereas those in light green indicate RuvC cleavage from within a RuvABC complex. Arrows in light blue indicate RuvAB helicase action, whereas that in pink indicates similar action by RecG or RecQ helicases. Black lines are unreplicated DNA, and red/pink lines newly replicated daughter strands. (a) Reannealing of daughter strands at a stalled replication fork (closed triangle indicates a nontemplate lesion on the leading strand). Reannealing can be mediated by RecG (18) and facilitated by positive supercoiling ahead of the fork (27). RuvAB and RecA may also promote the growth of the reversed fork once fork reversal has been initiated. Lagging strand synthesis is indicated as proceeding beyond the lesion, thereby providing an opportunity to replicate past the lesion by copying of the switched template in a reaction that uses recombination proteins, but not recombination (pink line; ref. 18). (b) Productive RuvAB branch migration to extend the four-way HJ (RuvB is cartooned as a pair of cylinders on opposed arms of the HJ). Note that RuvB binding to the other two arms of the junction (step l) will lead to abortion of the four-way junction by branch migration (step p). (c and m) Action of RuvABC to cleave the strands 3′ of the bound RuvB on the branch point side, to generate broken forks (corresponding to single strand lesions in the leading and lagging parental template strands respectively; ref. 24). RecABCD-mediated reinvasion of the broken ends leads to rebuilt replication forks that most readily yield noncrossover (d_–_f) and crossover (n) chromosomes, respectively. Note that after reinvasion, a further round of RuvABC action is required; in each case the “productive” orientation of RuvB binding gives the majority species shown (f and n). Binding of RuvB in the abortive configuration either will act to reverse the invasion or will lead to RuvC cleavage to give the minority products, dimers and monomers, respectively (k, o). (g–i). RecABCD-mediated invasion of the end created by fork reversal into its homologous region to generate a molecule containing two HJs (or a HJ and a three-way junction). Such an intermediate can be processed by crossover (i) or noncrossover (h) pathways by several ways that involve the simultaneous or sequential action of proteins at each of the branch points; we predict dimers to predominate over monomers in these events. (j) Processing of the reversed replication fork intermediate by RecG or RecQ helicases (equivalent to step p, promoted by RuvAB).

Figure 2

Figure 2

Alignment of E. coli DnaX, YcaJ, and RuvB (

clustal x

multiple sequence alignment program, v. 1.8,

http://www.ebi.ac.uk/clustalw/

). The sequences of YcaJ, RuvB, and DnaX all contain well-conserved nucleotide-binding sites with Walker A (GxxxxGKT/S) and Walker B (Dexx) motifs. The Zn-binding motif of DnaX is absent in YcaJ, but the putative ATPase sensor motifs (29) are present. Colors represent types of amino acids.

Figure 3

Figure 3

(A) An outline of the Xer recombination reaction. XerCD bind cooperatively at dif, psi, or cer recombination sites, ensuring synapsis (with the help of accessory sequences and proteins in the case of psi/cer) (i). XerC initiates catalysis (ii) to form a HJ intermediate, which undergoes a conformational change (iii) to provide a substrate for catalysis by XerD, which can then complete the recombination reaction (iv). There is normally a barrier to this conformational change, and XerC frequently catalyzes the conversion of the HJ back to substrate (ii). In recombination at psi, the proteins PepA and ArgA-P facilitate the HJ conformational change, whereas in recombination at dif, FtsKc is thought to facilitate this change (34, 35). (B, C) Species specificity of FtsK action. FtsKformula image cells (DS9041) were transformed with pBAD expression vectors (48) carrying full-length FtsK proteins (B) or the C-terminal domains (C) of different species. To assay for Xer recombination, they were transformed with a plasmid containing two dif sites and grown in conditions of repression (−; 0.2% glucose) or induction (+; 0.2% arabinose) of the expression vectors (34). Induction was checked by Western blot analysis by using an antibody directed against a FLAG epitope fused to the N termini of the constructs, after resolution of the protein extracts on a 6% (B) or an 8% (C) SDS/PAGE. (D) Alignment of the C-terminal domains of FtsK homologues. Identical residues are indicated by stars, conservative substitutions by dots. Open boxes underline regions predicted to adopt an α-helix conformation by

predictprotein phd

software v. 1.96,

http://www.embl-heidelberg.de/predictprotein/predictprotein.html

whereas black arrows underline those predicted to form β sheets. Ec: E. coli FtsK, Hi: H. influenzae FtsK and Bs: B. subtilis SpoIIIE.

Figure 4

Figure 4

The C-terminal domain of FtsK is randomly distributed throughout the cytoplasm. FtsKformula image cells (DS9041) were transformed with pBAD expression vectors carrying an N-terminal fusion of the green fluorescent protein (GFP) to full-length FtsK or to the C-terminal domain of FtsK (FtsKc). Cells were grown to midexponential phase in LB supplemented with 0.1% (full-length) or 0.2% (FtsKc) arabinose. Nucleoids were stained by using 4′,6-diamidino-2-phenylindole (DAPI). Phase-contrast and fluorescent images were acquired by using a cooled charge-coupled device camera (Princeton Instruments, Trenton, NJ) and

metamorph image acquisition

software (Universal Imaging, Media, PA) from an Olympus BX50 (New Hyde Park, NY) fluorescence microscope. Shown are overlays of the DAPI image in red and the GFP image in green. Full-length FtsK frequently localizes to the septum, whereas FtsKc is always distributed throughout the cytosol and never found at the septum.

Figure 5

Figure 5

A model for chromosome segregation in E. coli. In contrast to the replication origins (large dark gray circles), the replication terminus region, which contains the dif site (open triangle), stays localized at midcell (49). As a consequence, sister dif sites can be synapsed by the XerCD recombinases (small white and light gray circles) whether the chromosomes form a dimer or not. This leads to cycles of HJ formation and resolution by XerC. If the chromosomes are monomeric, segregation will eventually break the synaptic complex and move the sites away from midcell before septum closure. If the chromosomes form a dimer, the synaptic complex will stay trapped at midcell, which allows access to FtsK. FtsK mediates the HJ conformation change needed to activate catalysis by XerD, thus coordinating resolution of chromosome dimers to cell division.

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