Search

Transposons have had a pivotal role in genome evolution.sup.1 and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase.sup.2, an essential component of the adaptive immune system in jawed vertebrates.sup.3. Here we report one crystal structure and five cryo-electron microscopy structures of Transib.sup.4,5, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0-4.6 Å. These structures reveal a butterfly-shaped complex that undergoes two cycles of marked conformational changes in which the 'wings' of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA and close again to capture and attack target DNA. Transib possesses unique structural elements that compensate for the absence of a RAG2 partner, including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help to control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the detailed reaction pathway of a eukaryotic cut-and-paste transposase and illuminate some of the earliest steps in the evolution of the RAG recombinase. Analysis of multiple structures of the Helicoverpa zea DNA transposase Transib, determined by X-ray crystallography and cryo-electron microscopy, reveals the detailed pathway of the transposition reaction and sheds light on the evolution of the RAG recombinase., Author(s): Chang Liu [sup.1] , Yang Yang [sup.2] [sup.3] , David G. Schatz [sup.1] Author Affiliations: (1) Department of Immunobiology, Yale School of Medicine, New Haven, USA (2) Department of [...]

Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis (1). There is growing evidence that cohesin also forms long-range chromosomal cis-interactions (2-4) and may regulate gene expression (2-10) in association with CTCF (8,9), mediator (4) or tissue-specific transcription factors (10). Human cohesin opathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions (7), but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila (11-13)--has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) a locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter-enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery (14,15) and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome (4,8-10), defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system., The somatic rearrangement of lymphocyte receptor loci is central to adaptive immunity16. Gene segments distributed over millions of base pairs of genomic DNA are transcribed, brought into proximity with each [...]

Somatic hypermutation introduces point mutations into immunoglobulin genes in germinal centre B cells during an immune response. The reaction is initiated by cytosine deamination by the activation-induced deaminase (AID) and [...]

Targeted hypermutation of immunoglobulin variable region genes occurs in B cells during an immune response (1), and gives rise to families of related mutant antibodies which are then selected for their binding affinity to the immunizing antigen (2). Somatic hypermutation predominantly generates point mutations, many of which occur at specific residues (hotspots) (3). The reaction has been linked to transcription and requires the presence of immunoglobulin enhancers (4-6), but replacement of the variable gene by heterologous sequences, or the variable region promoter by a heterologous promoter, does not interfere with the mutation process (7,8). Here we show the existence of abundant DNA double-strand breaks (DSBs) in hypermutating sequences. Generation of the DSBs is coupled to transcription, enhancer-dependent, and correlates with the appearance of nearby mutations. Furthermore, the DSBs are cell-cycle restricted, being found almost exclusively in cells that have completed, or nearly completed, DNA replication. We propose a model for somatic hypermutation in which mutations are introduced into the DNA during repair of DSBs by homologous recombination. The finding of DSBs during somatic hypermutation may help to explain the chromosomal translocations found in some B-cell tumours., Somatic hypermutation was originally hypothesized to be a two-step process, initiated by cleavage of the DNA within the mutating region, and subsequently resolved by error-prone repair (9). Direct evidence for [...]

A site-specific recombination reaction, known as V(D)j recombination, is responsible for assembling immunoglobulin and T-cell-receptor genes from gene segments. Recombination-activating genes, RAG1 and RAG2, encode proteins that are essential in this process. They are found in all jawed vertebrates but not in jawless vertebrates, suggesting that split antigen receptor genes arose with the divergence of bony and cartilaginous fish. New research shows that the repertoire of immunoglobins and T-cell receptors is owed to a single transposon insertion occurring some 450 million years ago.

In clonal selection in the adaptive immune response B and T cells have a diverse array of antigen receptors before encountering antigens. New studies have indicated that assembly of antigen-receptor genes in mature B cells, may be reactivated in response to antigen. Two research groups report a new tool that may enable a more sophisticated analysis of B and T cell development. One group used a large reporter transgene comprising RAG chromosomal regions with GFP substituted fro RAG2, while the other group created mice where the endogenous RAG2 locus directs expression of a RAG2-GFP fusion protein. Each group detected a subset of RAG-expressing B cells in the spleen.

Presents a report on the hypermutation of immunoglobulin variable region genes and on the existence of abundant DNA double-strand breaks (DSBs) in hypermutating sequences. How the hypermutation occurs in B cells during an immune response; Generation of the DSBs and how it is coupled to transcription, enhancer-dependent, and correlates with the appearance of nearby mutations; Proposition of a model for somatic hypermutation in which mutations are introduced into the DNA during repair of DSBs by homologous recombination.