Illustration courtesy of Anmei Little.
In the era of high-resolution imaging and advanced computer modeling, scientists can see a protein’s composition in great detail and gain extensive information about its properties and function. However, these images are only transient snapshots of the protein. To better understand protein structure and function, scientists have more recently developed powerful experimental and analytical tools can be applied to deduce a protein’s provenance and fill in the rest of the story. One such success story has been reported by the Schatz Lab at the Yale School of Medicine, which used cutting-edge imaging technology to characterize a Transib transposase protein to elucidate the evolutionary story of a critical enzyme in the vertebrate adaptive immune system.
DNA Recombination and Adaptive Immunity
David Schatz, Chair of Immunobiology and Professor of Immunobiology and Molecular Biophysics and Biochemistry at Yale, pioneered his line of research in the late 1980s as a graduate student at MIT studying V(D)J recombination, a specific type of DNA recombination event that only occurs in developing B and T cells, and is an integral part of the adaptive immune system. “Antibody genes and T cell receptor genes are in a disassembled nonfunctional state in the germ line chromosomes. Specifically, these genes are broken up into small pieces of DNA called V, D, and J and need to be brought together by cutting and recombining the chromosome,” Schatz said. Although the existence of this type of recombination was widely accepted in the 1980s, the nature of the biomolecules carrying out this process remained elusive.
To discover the genes responsible for the recombination, Schatz took cells and transferred large segments of chromosomal DNA into them to see which combination of DNAs would yield the recombination reaction. After extensive experimentation, Schatz discovered two genes: RAG1 and RAG2, which together encode the RAG1-RAG2 recombinase, an enzyme that facilitates the cutting of the V, D, and J DNA segments in V(D)J recombination. “It was a major advancement for the field,” Schatz said. “Once they were isolated, they provided the critical tools for studying the reaction and its regulation.”
In Vitro but not In Vivo
In 1998, Schatz and his research team made a surprising discovery: recombinant RAG1-RAG2 recombinase was able to perform transposition of its encoding genes in vitro. This immediately raised the question: if this transposition reaction can occur in a test tube, does it also occur in the human body? It doesn’t, the lab quickly discovered, and Schatz and his team spent the next twenty years trying to learn why. However, this discovery supported another theory that is now well-accepted: the precursors of these recombination activating genes (RAG) were transposons, which Schatz describes to be “rather selfish genetic elements,” whose main functions are to encode the machinery necessary for replicating and translocating themselves.
By studying the structure of Transib, a RAG1-like transposase that the lab used as a proxy for how predecessors of the RAG1-RAG2 recombinase, the lab hoped to characterize the evolutionary predecessor of the recombinase, and discover how contemporary recombinase evolved to inhibit their transposase activity in vivo. Transib transposition and V(D)J recombination begin in a similar fashion to RAG1-RAG2 recombination. Either end of a terminal inverted repeated (TIR), a pattern of nucleotide bases which defines the transposon region, is nicked and excised. While in classic cut-and-paste transposition the Transib transposase facilitates the integration of the excised fragment into another region of the genome, in V(D)J recombination facilitated by RAG1-RAG2 recombinase, the two ends of the excised DNA fragment are ligated together and form a circular piece of DNA.
Transib Transposition Snapshots
Chang Liu of the Schatz lab and Yang Yang used X-ray crystallography and cryo-electron microscopy, high-resolution imaging techniques, to either crystallize protein and associated DNA or to freeze it to cryogenic temperatures, respectively, before imaging using electron microscopy techniques. The research team imaged at multiple stages in the Transib transposition reaction. Through this series of high resolution “snapshots,” the lab was able to piece together a highly dynamic 3D model of this transposition reaction with a level of detail and accuracy that has not been done for any other such reaction before this point.
Binding of the transposase Transib to its TIR DNA sequence causes the protein to adopt a conformation that resembles a butterfly with its wings extended with the DNA in the position of the butterfly’s antennas. The ‘wings’ of the enzyme fold inward as the DNA is cleaved, after which the wings then re-open. With this clear understanding of the Transib transposition mechanism, Schatz, Liu, and Yang were then able to compare their proxy for the precursor for the RAG1 subunit, which contains the active site of the enzyme complex and the DNA recognition sequence, of RAG1-RAG2 recombinase. Through this comparison between the two mechanisms provided, relevant information as to the evolution of the recombinase can be discovered.
Evolution of RAG1 and Transib
Structurally, RAG1 and Transib transposase are highly similar. The most notable differences between the two enzymes arise as a consequence of the respective presence or absence of RAG2. For instance, RAG1 possesses extra motifs that are the binding site for the RAG2 peptide. During formation of the enzyme-DNA complex, the RAG1-RAG2 recombinase experiences less drastic rotations of its chains than the Transib enzyme as RAG2 interacts with the DNA, forming stabilizing associations. RAG2 subsequently stabilizes the clamped conformation of the enzyme about the DNA, whereas Transib requires other factors to facilitate the same effect. Further, unlike the RAG1-RAG2 recombinase, Transib interacts with the ends of excised DNA, prohibiting the DNA from ligating to itself as in RAG1-RAG2 recombinase mediated V(D)J recombination.
The most significant difference between the two enzymes is that Transib has a critical target site-interaction loop that is missing in the recombinase due to the presence of the RAG2 subunit that renders this loop obsolete. This and other findings have led to the hypothesis that, at some point in evolutionary history, a Transib-like transposon gained the RAG2 gene, which would eventually developed into the first RAG1-RAG2 transposon.
Twenty years after the discovery of the transposition properties of RAG recombinase in vitro, Schatz and his research team pinpointed key amino acids and protein domains whose loss or gain drives the lack of transposition of RAG recombinase in cells using this concept of evolutionary genetics. Schatz has dedicated his thirty-year career to researching the mechanism of V(D)J recombination and the RAG recombinase. “We can now, in a detailed molecular and structural manner, tell the story of the evolutionary trajectory of the RAG system to the present day. While there are still some gaps in the narrative, there is a sense of satisfaction in having taken it this far,” Schatz said.
About the Author
Britt Bistis is a junior majoring in Molecular Biophysics and Biochemistry. She works in the Noonan lab working to characterize how mutations in high-confidence autism risk genes alter the developmental trajectory of the brain and the regulatory mechanisms through which this may occur. Outside of the lab, she can be found doing volunteer work in programs for special needs students and science outreach programs or horseback riding.
The author would like to thank Professor David Schatz for his time and commitment to his research.
Liu, C., Yang, Y., & Schatz, D. G. (2019). Structures of a RAG-like transposase during cut-and-paste transposition. Nature, 575, 540-544.
Huang, S., Tao, X., Yuan, S., Zhang, Y., Li, P., Beilinson, H. A., … Xu, A. (2016). Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination. Cell, 166, 102-14.
Carmona, L. M., Fugmann, S.D., & Schatz, D.G. (2016). Collaboration of RAG2 with RAG1-like proteins during the evolution of V(D)J recombination. Genes & Development, 30, 909-17.