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Eukaryon

The Fight Against Transgressing Transposons

Alejandro Rodríguez
Lake Forest College
Lake Forest, Illinois 60045

The protein Y-transposase mediates the excision and ligation of antibiotic resistant genes into a recipient bacterium’s genome. Structures of the enzyme in complex with DNA intermediates provide a better understanding of the underlying mechanism and spread of antibiotic resistance.

The discovery of penicillin in the early 20th century led to a tremendous decline in the number of deaths from antibiotic resistance bacteria. Since that discovery, antibiotic resistance has surpassed the effectiveness of available drugs; in fact, it was only in the 1980s when researchers found Enterococcus to be resistant to vancomycin, a third-line antibiotic (Falkham 1989). Following this, there has been a large increase in vancomycin-resistance, with vancomycin-resistant Enterococcus (VRE) causing up to 1,300 deaths per year (1). As a result, the issue of developing new antibiotics to combat this ever-growing problem seems to be a losing battle, though structural research within the past year now provides better insight into possible future treatments.

Much of our understanding of the problem has shifted to transposons, also known as “jumping genes.” Barbara McClintock discovered these genes in the 1950s when doing experiments on maize inheritance patterns and found that offspring had unexpected variation due to movement of genes responsible for patterning (2).

While these genes were identified in maize, they have also been observed in bacteria and were termed “conjugative transposons” (CTns) since they are transferred between bacterium. Each conjugative transposon is made of three components: inverted repeats, structural genes, and then the genes responsible for antibiotic resistance. The resistance genes likely contain information for the creation of peptides that shield the cell wall of the bacteria from antibiotics. As of now, many families of conjugative transposons have been identified, and the family Tn1549 has been found to provide resistance to vancomycin (3).

Though transposons may provide the change in phenotypic expression observed, researchers still did not know what the movement of these genes was due to. Now, this has been attributed to integrase, a transposase enzyme. Wozniack and Waldor (2010) provided a mechanism by which integrase may excise and ligate transposons into a recipient bacterium’s genome: 1) a circular intermediate (CI) is excised from the donor by recognition of inverted repeats 2) the CI is moved to the recipient through conjugation and the genes are duplicated and 3) the CI is integrated into the recipient’s genome (4).

Although Wozniack and Waldor’s research provided a mechanism for transposition, there remained a lack of structural data to support a stronger underlying mechanism. Rubio-Cosials et al. (2018) recently determined the structure of transposase in complex with CI to fill this gap in knowledge (5). Using a technique known as x-ray crystallography, they collected diffraction data from x-ray beams deflected from a crystallized protein to provide the first glimpse of this interaction. Through the structure, the authors identified many features. They found how the enzyme binds to DNA; where transposon end-recognition occurs; what position in the inverted repeats the enzyme cleaves; whether a Holliday junction intermediate can be resolved; and also reveal that a key structure known as alpha-M can be targeted by drugs to inhibit integrase activity, thereby illuminating on a more detailed mechanism lacking from Wozniack and Waldor’s proposed mechanism.  

Based on the observed crystal, the researchers discovered that integrase (Int) binds as a dimer to the CI, with each monomer identifying one inverted repeat of the gene. Inverted repeats are also known as palindromes because they can be read left to right in the same manner. Within the context of transposition, each integrase monomer is like a hand that identifies a palindrome, such as the word “racecar.” This allows for the enzyme to determine which positions to excise within the gene with incredible accuracy. To aid in the detection of these IR regions, the residue N150 of integrase forms a hydrophobic network with the bases preceding the cross-over region of the DNA (Figure 1). Residue of N150 integrase forms hydrophobic network with DNA bases.

In addition to the discovery of how transposon end recognition occurs, Rubio-Cosials et al. found another striking feature in the crystal structure with what they termed “base flipping.” Prior to excising the transposon from the rest of the DNA, the residue R153, located on a domain within integrase known as the core DNA binding domain (CB), will push the first base of the crossover region, with the same process occurring on the other side of the transposon. Mutagenesis studies on R153 demonstrated that without base flipping, the transposon remains intact, thus revealing that the unwinding of DNA by R153 is required for effective excision, though the authors do not prescribe an exact role for base flipping in the mechanism for transposition.

Prior to this study, one surprising feature of integrase was how it was seemingly able to excise varying lengths of transposons, regardless of their sequences (6). By using DNA intermediates of varying lengths, however, Rubio-Cosials et al. discovered that the enzyme interacts with each DNA variant similarly, with each protein unit “grabbing” the IR regions through base-specific contacts with similar affinity for each substrate. Interestingly, the authors show through the crystal structure that the flexibility created by the enzyme through base flipping is what allows sequences of varying lengths to be excised with minimal difficulty. As a result, the effectiveness of integrase in interacting with different sequences likely enhances its ability to confer antibiotic resistance to different strains of bacteria.

Furthermore, the authors answered the question of whether integrase could resolve a Holliday junction intermediate formed after the enzyme attempts to insert the transposon in the recipient bacterium’s genome. A Holliday junction intermediate is a 4-way double-stranded junction. The purpose of forming this junction can be either homologous recombination, which increases genetic diversity by exchanging DNA between strands, repairing double strand breaks by using an undamaged strand as a template, or site-specific recombination, which is a type of genetic recombination that uses enzymes known as recombinases to perform the DNA rearrangements. Typically, tyrosine recombinases form a tetramer complex instead of a dimeric complex, as seen with integrase. Rubio-Cosials et al. showed that by radiolabeling each DNA region, the IRL (left inverted repeat) and IRR (right inverted repeat), integrase cleaved and joined different segments of DNA together, thereby resolving the 4-way junction intermediate. This finding is important because it confirms integrase’s function as a site-specific recombinase capable of strand exchange.

Lastly, and perhaps most intriguing given its translational appeal, Rubio-Cosials et al. found that targeting the C-terminal helix of the dimer could be the key to fighting antibiotic resistance. The C-terminal helix is a structure important for binding both monomers together in the complex. Truncations or deletions of certain portions of the C-terminal helix in a region known as the alpha-M inhibited dimerization. After this discovery, the authors created a peptide to target the C-terminal region and their findings demonstrated that an increased dose prevented integrase activity. While this seems to be an incredible finding, the enzyme required a dose of 4mM before all of its activity was inhibited, indicating that more research is required to understand what regions of the enzyme should be targeted to increase the potency of drugs.

Given the results, it is important to acknowledge the role x-ray crystallography had in illuminating these findings. Wozniak and Waldor (2010) were unable to provide a detailed mechanism for transposition; however, the methods employed by Rubio-Cosials et al. illustrated many of the missing pieces. For example, the crystal structure determined by x-ray crystallography revealed the exact position of excision by integrase. In vivo sequencing previously led researchers to believe cleavage occurs at the border of the inverted repeat sequences (7). On the other hand, Rubio-Cosials et al. determined that cleavage occurs within the IR sequences. The crystal structure also revealed how N150 is required for transposon end recognition through its base-specific contacts and that base flipping exists prior to excision. X-ray crystallography allows for molecules with large molecular weights and protein-nucleic acid complexes to be captured in a higher resolution than other techniques, and the resolution of the integrase-CI complex at 2.7 angstroms confirms this. 

While the benefits of the x-ray crystal structure are evident, there were also many weaknesses. Each monomer contains three domains: a core DNA binding domain (CB), an arm-binding domain (AB) and a catalytic binding domain (CB). Rubio-Cosials et al. left out the entire AB domain from their determined structure since the AB domain remains too flexible to be captured. The disadvantages that come from this are realized when it is understood that in order to make a crystal, a variant of the protein must often be made to increase thermostability to increase the chance of crystallization, which may be one reason why the AB domain was removed, though the authors do not specify their reasoning in detail (8). In contrast, another technique known as cryo-EM that does not need a crystal and merely captures different states of a frozen molecule to develop a 3D model.

Ultimately, the research provided by Rubio-Cosials et al. gives incredible insight that could lead to  possible solutions for the growing problem of antibiotic resistance. What their research provides is a better understanding of how the enzyme that confers resistance recognizes DNA sequences, where it cleaves them, how it exchanges genetic information, and what structures within it are best suited for targeting by drugs. Although the authors do not propose future directions, it is evident that determining the crystal structures of other enzymes in the tyrosine recombinase family and comparing them with the newly developed mechanism for transposition could show whether this mechanism is special in the Tn1549 family or if it is shared among all CTn transposases. Additionally, only the initial steps of the mechanism, which include transposon end recognition and cleavage, have been crystallized, so future research should identify crystal structure capturing integrase resolving the HJ intermediate and inserting the transposon into the recipient bacterium’s genome. Whether this is possible at all remains to be seen.

 

References

  1. ar-threats-2013-508.pdf [Internet]. [cited 2019 Apr 26]. Available from: https://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf
  2. McClintock B. The origin and behavior of mutable loci in maize. PNAS. 1950 Jun 1;36(6):344–55. 
  3. van Hal SJ, Ip CLC, Ansari MA, Wilson DJ, Espedido BA, Jensen SO, et al. Evolutionary dynamics of Enterococcus faecium reveals complex genomic relationships between isolates with independent emergence of vancomycin resistance. Microb Genom [Internet]. 2016 Jan 19 [cited 2019 Apr 26];2(1). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5049587/
  4. Wozniak RAF, Waldor MK. Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow. Nature Reviews Microbiology. 2010 Aug;8(8):552–63. 
  5. Rubio-Cosials A, Schulz EC, Lambertsen L, Smyshlyaev G, Rojas-Cordova C, Forslund K, et al. Transposase-DNA Complex Structures Reveal Mechanisms for Conjugative Transposition of Antibiotic Resistance. Cell. 2018 Mar 22;173(1):208-220.e20. 
  6. Launay A, Ballard SA, Johnson PDR, Grayson ML, Lambert T. Transfer of Vancomycin Resistance Transposon Tn1549 from Clostridium symbiosum to Enterococcus spp. in the Gut of Gnotobiotic Mice. Antimicrob Agents Chemother. 2006 Mar;50(3):1054–62. 
  7. Conjugative transposition of the vancomycin resistance carrying Tn1549: enzymatic requirements and target site preferences - Lambertsen - 2018 - Molecular Microbiology - Wiley Online Library [Internet]. [cited 2019 Apr 26]. Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/mmi.13905
  8. Deller MC, Kong L, Rupp B. Protein stability: a crystallographer’s perspective. Acta Crystallogr F Struct Biol Commun. 2016 Jan 26;72(Pt 2):72–95. 

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