Biomedical subjects

Sheryl K Elkin

Publications and source records attributed to Sheryl K Elkin.

5 recordsLinked to original sources

PubMed2005

A PHD finger motif in the C terminus of RAG2 modulates recombination activity.

The RAG1 and RAG2 proteins catalyze V(D)J recombination and are essential for generation of the diverse repertoire of antigen receptor genes and effective immune responses. RAG2 is composed of a "core" domain that is required for the recombination reaction and a C-terminal nonessential or "non-core" region. Recent evidence has emerged arguing that the non-core region plays a critical regulatory role in the recombination reaction, and mutations in this region have been identified in patients with immunodeficiencies. Here we present the first structural data for the RAG2 protein, using NMR spectroscopy to demonstrate that the C terminus of RAG2 contains a noncanonical PHD finger. All of the non-core mutations of RAG2 that are implicated in the development of immunodeficiencies are located within the PHD finger, at either zinc-coordinating residues or residues adjacent to an alpha-helix on the surface of the domain that participates in binding to the signaling molecules, phosphoinositides. Functional analysis of disease and phosphoinositide-binding mutations reveals novel intramolecular interactions within the non-core region and suggests that the PHD finger adopts two distinct states. We propose a model in which the equilibrium between these states modulates recombination activity. Together, these data identify the PHD finger as a novel and functionally important domain of RAG2.

Amino Acid Sequence↗

PubMed2004

ATP-dependent remodeling by SWI/SNF and ISWI proteins stimulates V(D)J cleavage of 5 S arrays.

Control of V(D)J recombination is critical for the generation of a fully developed immune repertoire. The molecular mechanisms underlying the regulation of antigen receptor gene assembly are beginning to be revealed. Here we studied the influence of chromatin modifications on V(D)J cleavage of a polynucleosomal substrate, in which V(D)J cleavage is greatly reduced compared with naked DNA. ATP-dependent remodeling by human SWI/SNF (hSWI/SNF) in the presence of HMG1 led to a substantial increase of cleavage by the recombination activation gene (RAG) proteins. Either BRG1, the ATPase subunit of hSWI/SNF, or SNF2h, the ATPase of human ISWI complexes, was capable of stimulating V(D)J cleavage of the array, although these remodelers act by different mechanisms. No effect of histone hyperacetylation was detectable in this system. As is observed on naked DNA, in the presence of core RAG1, the full-length RAG2 protein proved to be more active than core RAG2 on these polynucleosomal arrays, reinforcing the importance of the RAG2 C-terminal domain for efficient recombination. Comparison of 5 S array cleavage by the RAG proteins or by the restriction enzyme HhaI after remodeling by hSWI/SNF suggested that RAG proteins and HhaI might have different requirements for maximal accessibility of the substrate.

Adenosine Triphosphatases↗

PubMed2004

Ordered DNA release and target capture in RAG transposition.

Following V(D)J cleavage, the newly liberated DNA signal ends can be either fused together into a signal joint or used as donor DNA in RAG-mediated transposition. We find that both V(D)J cleavage and release of flanking coding DNA occur before the target capture step of transposition can proceed; no coding DNA is ever detected in the target capture complex. Separately from its role in V(D)J cleavage, the DDE motif of the RAG1/2 active site is specifically required for target DNA capture. The requirement for cleavage and release of coding DNA prior to either physical target binding or functional target commitment suggests that the RAG1/2 transposase contains a single binding site for non-RSS DNA that can accommodate either target DNA or coding DNA, but not both together. Perhaps the presence of coding DNA may aid in preventing transpositional resolution of V(D)J recombination intermediates.

Binding Sites↗

PubMed2003

The C-terminal portion of RAG2 protects against transposition in vitro.

The assembly of antigen receptor genes by V(D)J recombination is initiated by the RAG1/RAG2 protein complex, which introduces double-strand breaks between recombination signal sequences and their coding DNA. Truncated forms of RAG1 and RAG2 are functional in vivo and have been used to study V(D)J cleavage, hybrid joint formation and transposition in vitro. Here we have characterized the activities of the full-length proteins. Unlike core RAG2, which supports robust transposition in vitro, full-length RAG2 blocks transposition of signal ends following V(D)J cleavage. Thus, one role of this non-catalytic domain may be to prevent transposition in developing lymphoid cells. Although full-length RAG1 and RAG2 proteins rarely form hybrid joints in vivo in the absence of non-homologous end-joining factors, we show that the full-length proteins alone can catalyze this reaction in vitro.

DNA↗

PubMed2003

Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice.

The recombination-activating gene (RAG)1 and RAG2 proteins comprise the lymphocyte-specific components of the V(D)J recombinase and are required for the assembly of antigen-receptor variable-region genes. A mutant truncated RAG2 protein ("core" RAG2) lacking the C-terminal 144 amino acids, together with core RAG1, is able to mediate the basic biochemical steps required for V(D)J recombination in vitro and in transfected cell lines. Here we examine the effect of replacing the endogenous RAG2 locus in mice with core RAG2. These mice generate substantial numbers of B and T cells, demonstrating that the core RAG2 protein retains significant in vivo function. However, core RAG2 mice display a reduction in the total number of B and T cells, reflecting impaired lymphocyte development at the progenitor stage associated with reduced chromosomal V(D)J recombination. We discuss potential roles of the RAG2 C terminus in mediating rearrangement of endogenous antigen-receptor loci.

Animals↗