General public abstract:
Antibiotic resistance is a broad topic of societal and scientific importance. Reports of major nosocomial outbreaks and contaminations by drug resistanct bacteria have now become commonplace. One of the routes for the acquisition of bacterial resistance to antibiotics is the capture of resistance factors in the form of DNA molecules that encode an enzyme, or other transporter-like molecule, able to inactivate or expel the drug. We have focused our study on the mechanism of capture of DNA by bacteria, and its integration into the genome. This mechanism employs an enzyme called a recombinase that is able to bind, cut and paste pre-existing or new incoming DNA.
In order to study this enzyme, we have decided to use a multidisciplinary approach using biochemistry and structural biology, namely the method termed x-ray crystallography. In the first approach using biophysical tools we study the biochemical properties of both the recombinase and its target, the DNA molecule.
Site specific recombination impacts a wide variety of vital cellular processes ranging from recombination, repair and replication of DNA. Among the enzymes catalyzing the reaction are two prominent groups termed Tyrosine and Serine site specific recombinases. They use short palindromic sequences (30-40 base pairs) to perform DNA recombination. These two recombinase families differ by their active site residues, but also by their reaction mechanism and products. The Serine recombinases use an active site serine amino acid to perform the cleavage reaction and produce double strand breaks.
Tyrosine recombinases (Y-SSR) perform sequential DNA strand breaks with active site tyrosine residues, leading after the first cut to a four-way branched intermediate termed a Holliday junction. The integrons are gene platform in bacteria used for the capture and exchange of open-reading frames implicated in antibiotic resistance. The integron is composed of a promoter, insertion attI site, and several gene cassettes bearing an attC site and an open reading frame coding for resistance markers. The incoming cassettes are recombined at the attI and bring in new functions. Unlike classical tyrosine recombinases, integron Y-SSRs use a refolded single stranded DNA as substrate. Four recombinase molecules bind to two DNA half sites to form a recombination synapse. After the first cut by only two of the active recombinases molecules in the reaction synapse, and strand exchange, the synapse waits for the replication fork in order to resolve the Holliday junction and integrate the cassette. The key step in the reaction is the formation of the Holliday junction. This step determines whether the reaction should proced forward to yield recombinant products or reverse to give back substrates. The pause of the reaction is also physically encoded in the substrate itself. Indeed, extra_helical bases protrude upon folding of the single stranded DNA substrate. These bases play a critical role in the binding reaction, but also serve as mechanical barriers that prevent the inactive molecules to cleave the DNA.
The details of the reaction based on structural as well as biochemical support will help to design future biochemical pathways and new reagents to interfere with antibiotic resistance.