Thermal dependency of RAG1 self-association properties

<p>Abstract</p> <p>Background</p> <p>Functional immunoglobulin and T cell receptor genes are produced in developing lymphocytes by V(D)J recombination. The initial site-specific DNA cleavage steps in this process are catalyzed by the V(D)J recombinase, consisting of RAG1 and RAG2, which is directed to appropriate DNA cleavage sites by recognition of the conserved recombination signal sequence (RSS). RAG1 contains both the active site and the RSS binding domains, although RAG2 is also required for DNA cleavage activity. An understanding of the physicochemical properties of the RAG proteins, their association, and their interaction with the RSS is not yet well developed.</p> <p>Results</p> <p>Here, we further our investigations into the self-association properties of RAG1 by demonstrating that despite the presence of multiple RAG1 oligomers, only the dimeric form maintains the ability to interact with RAG2 and the RSS. However, facile aggregation of the dimeric form at physiological temperature may render this protein inactive in the absence of RAG2. Upon addition of RAG2 at 37°C, the preferentially stabilized V(D)J recombinase:RSS complex contains a single dimer of RAG1.</p> <p>Conclusion</p> <p>Together these results confirm that the functional form of RAG1 in V(D)J recombination is in the dimeric state, and that its stability under physiological conditions likely requires complex formation with RAG2. Additionally, in future structural and functional studies of RAG1, it will be important to take into account the temperature-dependent self-association properties of RAG1 described in this study.</p

Distinct roles for E12 and E47 in B cell specification and the sequential rearrangement of immunoglobulin light chain loci

The E2A gene products, E12 and E47, are critical regulators of B cell development. However, it remains elusive whether E12 and E47 have overlapping and/or distinct functions during B lymphopoiesis. We have generated mice deficient for either E12 or E47 and examined their roles in B cell maturation. We show that E47 is essential for developmental progression at the prepro–B cell stage, whereas E12 is dispensable for early B cell development, commitment, and maintenance. In contrast, both E12 and E47 play critical roles in pre–B and immature B cells to promote immunoglobulin λ (Igλ) germline transcription as well as Igλ VJ gene rearrangement. Furthermore, we show that E12 as well as E47 is required to promote receptor editing upon exposure to self-antigen. We demonstrate that increasing levels of E12 and E47 act to induce Igλ germline transcription, promote trimethylated lysine 4 on histone 3 (H3) as well as H3 acetylation across the Jλ region, and activate Igλ VJ gene rearrangement. We propose that in the pre–B and immature B cell compartments, gradients of E12 and E47 activities are established to mechanistically regulate the sequential rearrangement of the Ig light chain genes

DNA Cleavage Activity of the V(D)J Recombination Protein RAG1 Is Autoregulated

RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone. Furthermore, the catalytic activity of this domain on ss DNA is abolished by addition of the C-terminal domain of RAG1. The inhibitory effects of this latter domain are suppressed on substrates containing double-stranded (ds) DNA. Together, the activities of the reconstituted domains on ss versus mixed ds-ss DNA approximate the activity of intact RAG1 in the presence of RAG2. We propose how the combined actions of the RAG1 domains may function in V(D)J recombination and also in aberrant cleavage reactions that may lead to genomic instability in B and T lymphocytes

Analyses of temperature and temporal dependent aggregation of dimeric MCR1

SEC elution profiles of Fraction A, including material eluting in the void volume (V) after incubation for 30 min at 10, 25, and 37°C. The fraction of MCR1 (Fraction A) remaining in the dimeric state after incubation at 0, 10, 30, or 150 min at either 25 or 30°C, as determined by peak integration of SEC elution profiles. Fraction A sample for the 0 min time point was at 4°C prior to SEC analysis.<p><b>Copyright information:</b></p><p>Taken from "Thermal dependency of RAG1 self-association properties"</p><p>http://www.biomedcentral.com/1471-2091/9/5</p><p>BMC Biochemistry 2008;9():5-5.</p><p>Published online 30 Jan 2008</p><p>PMCID:PMC2270275.</p><p></p