Question

“In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH) and its cognate dif site (difH). Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE) in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis.”

article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC332523

What is one intrinsic problem with circular DNA molecules during homologous recombination? How does this effect cell division?

Bacteria with circular chromosomes typically contain site-specific tyrosine Xer recombinases and dif sites. Why are these recombinases and dif sites important to the bacterial cell?

Briefly explain how the XerC and XerD recombinases function in E.coli.

How does the H.pylori Xer recombinase system differ from the E.coli system? How is it similar?

E.coli typically removes topological links between concatenated DNA by using topoisomerase IV. H. pylori seems to lack topoisomerase IV. If this is the case, how does H. pylori deal with circular DNA dimers and/or multimers?

Answer 1

Homologous recombination is a type of genetic recombination procedure which involves exchange of nucleotide sequences between two similar or identical DNA molecules, which are part of a homologous pair of chromosomes. The process involves the cutting and joining of DNA sequences, wherein the nucleotide sequence of one strand is transferred onto another strand. Homologous recombination is carried out by cells in order to repair strand breaks in the DNA molecules and also create genetic variation.

Bacterial cells often contain extrachromosomal DNA in their cytoplasm, known as plasmids, which are covalently closed circular DNA molecules. In case of a homologous recombination event involving two cirular DNA molecules, recombination can lead to the formation of concatenated or attached chromosomes, which would evidently affect the viability of a bacterial cell. This concatenation of chromosomes pose a problem during cell division since the chromosomes are not properly segregated during cell division. Improper segregation of chromosomes due to dimer formation can lead to the inheritance of unequal chromosomes into daughter cells, leading to increased number of DNA molecules in a cell or aneuploidy.

As stated above, the process of homologous recombination between circular DNA molecules can generate concatenated chromosomes, which are detrimental for the bacterial cell. In order to resolve this problem, bacteria contain a site-specific recombination system involving a pair of tyrosine recombinases, XerC and XerD, which help in resolving the concatenated chromosomes. Xer C and Xer D recombinases along with the DNA translocase enzyme FtsK (Filamentation temperture sensititve gene K) bring about deconcatenation of chromosomal dimers in specific chromosomal sites known as 'dif' sites'. Thus the Xer/dif recombinase and resolution sites are important for bacterial cells because they lead to proper resolution of their circular DNA and effective segregation of chromosomes during cell division.

E. coli is a kind of bacterial with circular DNA, wherein homologous recombination leads to the formation of chromosome dimers, which pose a problem in chromosomal segregation during cell division. That's why the E. coli possess a XerC/D recombinase system which ensures that the E. coli chromosomal circular replicons are maintained in the monomeric state. The Xer recombinases act as site-specific topoisomerase I enzymes which unwind and relax the helix in plasmid regions that include a 'dif' site. The dif locus is a 28 nucleotide long palindromic motif with two inverted repeats separated by a central hexanucleotide, and the two inverted repeats are each attacked by eithe rpof the two Xer recombinases. these recombinases then produce negative tension in the supercoiled strands, thus resolving the problem of chromosome concatenation. (A 'dif' site stands for 'deletion-induced filamentation' which is a phenotype observed in E. coli starins deficient in Xer c, Xer D recombinase sor dif sites.)

In the gastric pathogenic bacterial Helicobacter pylori, there is only one Xer recombinase (Xer H) and its congante dif site (difH) , unlike the E. coli which has a pair of Xer recombinases. However, the mechanism of action of the Xer H recombinase and its effect on the cell division machinery of H. pylori was found to be the same as that of the Xer C/Xer D recombinases in E. coli. The Xer H recombinase was shown to function like topoisomerases and have deconcatenating effects in the H. pylori circular DNA molecules, aiding proper segregation during cell division. The mutation or absence of the Xer H recombinase gene in Helicobacter pylori showed an increase in the number of DNA molecules in the daughter cells of H. pylori, pointing to the fact that dimerization of chromosomes and resolving of recombination junctions could not be carried out in the absence of the Xer H recombinase.