Chapter 6
Topoisomerase IV is the main chromosome decatenase of E. coli and most bacteria [ 1 ] . 
Topo IV is a heterotetramer formed by dimers of ParC and ParE subunits [ 2 ] which present a high degree of structural homology with the GyrA and GyrB subunits of DNA gyrase , respectively . 
Alteration of Topo IV leads to severe chromosome segregation defects [ 2 ] but do not halt chromosome replication [ 3 ] . 
Recently a role for Topo IV in the segregation of replicating sister chromatids has been demonstrated [ 3 , 4 ] . 
It is therefore proposed that Topo IV works behind replication forks to remove precatenation links [ 5 , 6 ] that are formed by the rotation of the replication fork when DNA gyrase can not eliminate the positive superhelical tension generated by replication . 
Sister chromatids segregation is not a homogeneous process in E. coli , some regions of the chromosome appear to segregate a long time after their replication while some others segregate within minutes following their replication . 
Among the late segregation regions is the SNAPs regions that are enriched for GATC sequences that might recruit high amounts of SeqA protein that would inhibits Topo IV 
[ 7 ] . 
The terminus region of the chromosome also exhibits late segregation due to a combination of events : ( i ) the MatP-septal ring interaction [ 8 ] ; ( ii ) the MatP-MukB interaction and ( iii ) the MukB-Topo IV interaction [ 9 ] . 
These observations suggested that the decatenation activity of Topo IV is highly regulated in time and space . 
This is in good agreement with observations that Topo IV works preferentially late in the cell cycle [ 10 ] and in the chromosome terminus region at the dif site [ 12 ] . 
To get more insight into Topo IV regulation , we performed whole genome analysis of Topo IV binding and cleavage activity [ 11 ] . 
Topo IV has access to most of the genomic regions of E. coli but only selectively cleaves distinct genomic regions . 
Among the cleaved sites is the dif site which is by far the strongeest , confirming that for almost every cell cycle , decatenation events take place at dif on fully replicated chromosomes . 
To verify these observations we performed ChIP seq experiments and developed a new Topo IV-DNA co-immunoprecipitation method aimed at trapping only active Topo IV ( which we called NorfliP ) . 
These two methods are described in the present protocol . 
All solutions must be prepared using ultrapure water ( by purifying deionized water , to attain a sensitivity of 18 MΩ-cm at 25 C ) . 
Prepare the following buffers and stock solutions . 
Unless otherwise specified , filter solutions using a 0.2 μm low protein binding nonpyrogenic membranes . 
3 Methods
The following steps should be carried out at 4 C unless otherwise indicated . 
8 . 
Centrifuge beads for 30 s at 8000 g and recover the Topo IV-DNA complex found in the supernatant ( IP sample ) . 
9 . 
The ChIP-seq IP and input samples are de-cross-linked and proteins are degraded overnight at 65 C with 1 mg/mL proteinase K . 
The NorflIP IP and input samples are treated overnight at 65 C with 1 mg/mL proteinase K and 1 % SDS to degrade Topo IV covalently linked to the 50 end of DNA at the cleavage site . 
10 . 
Add 0.2 mg/mL RNAse A and incubate for 30 min at 37 C. 11 . 
Purify IP and input samples with a DNA cleanup kit and elute in 30 μL of Milli-Q water . 
Alternatively , the Mini - elute kit from Quiagen can be used to limit DNA loss during the cleaning process . 
12 . 
Measure DNA quality and quantity using Qubit dsDNA HS kit ( see Note 3 ) . 
13 . 
IP/input enrichment can then be preliminarily tested by qPCR using dif and gapA probes . 
Libraries were prepared according to Illumina 's instructions accompanying the DNA Sample Kit ( FC-104-5001 ) . 
Briefly , DNA was end-repaired using a combination of T4 DNA polymer-ase , E. coli DNA Pol I large fragment ( Klenow polymerase ) and T4 polynucleotide kinase . 
The blunt , phosphorylated ends were trea-ted with Klenow fragment ( 30 to 50 exo minus ) and dATP to yield a protruding 3 - ` A ' base for ligation of Illumina 's adapters which have a single ` T ' base overhang at the 30 end . 
After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~ 250 bp ( insert plus adaptor and PCR primer sequences ) were band isolated from an agarose gel . 
The purified DNA was captured on an Illumina flow cell for cluster generation . 
Libraries were sequenced on the Genome Analyzer following the manufacturer 's protocols with single read for 50 cycles . 
Sequencing results were processed by the IMAGIF facility . 
Base calls were performed using CASAVA version 1.8.2 . 
ChIP-seq and NorflIP reads were aligned to the E. coli NC_000913 genome using BWA 0.6.2 . 
A custom made pipeline for the analysis of sequencing data was developed with Matlab ( available upon request ) . 
Briefly , the number of reads for the input and IP data was smoothed over a 200 bp window . 
Forward and reverse signals were added , reads were normalized to the total number of reads in each experiment , strong nonspecific signals observed in unrelated experiments were removed , data were exported to the UCSC genome browser ( http://archaea.ucsc.edu ) for visualization and comparisons 
Several highly-enriched sites were observed in the IP samples . 
Interestingly one of these sites corresponds to the dif site ( position 1.58 Mb ) , which has previously been identified as a strong Topo IV cleavage site in the presence of norfloxacin [ 12 ] . 
We also observed strong enrichment over rRNA operons , tRNA and IS sequences . 
To address the significance of the enrichment at rRNA , tRNA , and IS , we monitored these sites in ChIP-seq experiments performed in the same conditions with a MatP-flag strain and mock IP performed with strain that did not contain any flag tagged protein . 
Both MatP and Mock IP presented significant signals on rRNA , tRNA , and IS loci . 
This observation suggested that Topo IV enrichment at rRNA , tRNAs and IS was an artifact of the ChIP-Seq technique . 
By contrast no enrichment was observed at the dif site in the MatP and mock-IP experiments , we therefore considered dif to be a genuine Topo IV binding site and compared every enriched region ( > 2 fold ) with the dif IP . 
We filtered the raw data for regions presenting the highest Pearson correlation with the dif signa 
4 Notes
( P > 0.7 ) . 
This procedure discarded many highly enriched regions . 
An example of a site presenting a selected Topo IV IP/input signal suggesting a specific binding is presented on Fig. 1 ( red graphs ) . 
The strongest IP/input ratio was observed at dif and a locus close to the yebV gene ( 1.9 Mb ) . 
They present a characteristic shape ( Fig. 1 blue graphs , see Note 4 ) that allows the automatic detection of lower amplitude peaks but preserving the characteristic shape . 
We measured Pearson correlation coefficient with the dif and the yebV site for 600 bp sliding windows over the entire genome . 
Peaks with a Pearson correlation above 0.7 were considered as putative Topo IV cleavage sites . 
Interestingly in the NorflIP experiments nonspecific signal was observed over rRNA and IS regions but not on tRNA . 
This suggested that immunoprecipitation signals over tRNA are artefacts linked to formaldehyde but not to the Flag immunoprecipitation .