The RAG1 and RAG2 proteins initiate V(D)J recombination by introducing double-strand breaks at the border between a recombination signal sequence (RSS) and a coding segment. To understand the distinct functions of RAG1 and RAG2 in signal recognition, we have compared the DNA binding activities of RAG1 alone and RAG1 plus RAG2 by gel retardation and footprinting analyses. RAG1 exhibits only a three-to fivefold preference for binding DNA containing an RSS over random sequence DNA. Although direct binding of RAG2 by itself was not detected, the presence of both RAG1 and RAG2 results in the formation of a RAG1-RAG2-DNA complex which is more stable and more specific than the RAG1-DNA complex and is active in V(D)J cleavage. These results suggest that biologically effective discrimination between an RSS and nonspecific sequences requires both RAG1 and RAG2. Unlike the binding of RAG1 plus RAG2, RAG1 can bind to DNA in the absence of a divalent metal ion and does not require the presence of coding flank sequence. Footprinting of the RAG1-RAG2 complex with 1,10-phenanthroline-copper and dimethyl sulfate protection reveal that both the heptamer and the nonamer are involved. The nonamer is protected, with extensive protein contacts within the minor groove. Conversely, the heptamer is rendered more accessible to chemical attack, suggesting that binding of RAG1 plus RAG2 distorts the DNA near the coding/signal border.Functional immunoglobulin and T-cell receptor genes are assembled in early B-or T-cell development by recombination events collectively termed V(D)J recombination (18,36). The V, D, and J gene segments are flanked by recombination signal sequences (RSS) that are composed of highly conserved heptamer and nonamer motifs separated by a relatively nonconserved spacer of 12 or 23 bp (2, 11). All segments of one class (V, D, or J) are flanked by RSS of the same spacer length. Recombination preferentially takes place between RSS of different spacer lengths (the "12/23 rule"), thus directing assembly of functionally relevant gene segments (18).The recombination reaction can be divided into two stages; first, double-strand breaks (DSB) are created at the border of the RSS and coding segment. Broken coding ends are covalently sealed in a hairpin structure while signal ends are blunt, 5Ј-phosphorylated molecules (25,26,31). In the second stage, the broken molecules are processed and ligated to form signal and coding joints. Signal joints are formed by precise ligation of signal ends in a head-to-head fashion. Coding ends are ligated imprecisely to form coding joints, thus introducing junctional diversity. The first stage of recombination is mediated by the lymphoid-specific genes RAG1 and RAG2 (22, 30). The later stages of the reaction require a number of factors, including many involved in DSB repair (14).The RAG proteins together carry out the same cleavage reaction in vitro as is observed in vivo, introducing a DSB at the coding/signal border (20). This cleavage is generated in two steps. First, a nick is introduced at...
The mouse transcription factor PEBP2 is a heterodimer of two subunits: a DNA binding subunit ␣ and its partner subunit . The ␣ subunit shares a region of high homology, termed the Runt domain, with the products of the Drosophila melanogaster segmentation gene runt and the human acute myeloid leukemia-related gene AML1. To study the molecular basis for the DNA binding and heterodimerization functions of this factor, we constructed series of deletions of the ␣ and  subunits and examined their activities by electrophoretic mobility shift and affinity column assays. The minimal functional region of the ␣ subunit for DNA binding and dimerization was shown to coincide with the Runt domain. On the other hand, the region of the  subunit required for heterodimerization was localized to the Nterminal 135 amino acids. Furthermore, it was found that the DNA binding activity of the Runt domain is regulated by a reduction/oxidization (redox) mechanism and that its reductively activated state, which is extremely labile, is stabilized by the  subunit. These findings add a new layer to the mechanism and significance of the regulatory interplay between the two subunits of PEBP2.
During lymphocyte development, V(D)J recombination is highly regulated in the contexts of lineage specificity, developmental-stage specificity, and allelic exclusion (4, 5). In addition, developing lymphocytes use strategies that require productive V(D)J rearrangements for continued developmental progression (4, 5). For example, Ig heavy chain (IgH) variable-region genes are assembled in progenitor (pro) B cells via an ordered process in which D H -to-J H rearrangement occurs on both alleles before V H -to-DJ H rearrangement. Productive V H -to-DJ H rearrangements produce cell-surface expression of IgH chains that signal expansion and differentiation of pro-B cells to the pre-B cell stage. This expression of IgH chains also signals the cessation of further V H -to-DJ H rearrangement via feedback regulation. Pro-B cells that first assemble nonproductive V H -to-DJ H rearrangements proceed to rearrange their second allele.Both RAG1 and RAG2 are required for V(D)J recombination, because RAG1-or RAG2-deficient mice exhibit a complete block in lymphocyte development at the progenitor stage (6, 7). In addition, RAG1 and RAG2 together are sufficient to initiate V(D)J recombination in vitro (8). Nearly all in vitro studies of RAG function have used the minimal regions of RAG1 (amino acids 384-1,008 of 1,040) and RAG2 (amino acids 1-383 of 527) required for activity, because full-length RAG1 and RAG2 are largely insoluble. However, the activities of these mutant ''core'' proteins differ from those of the full-length RAGs when assayed with extrachromosomal substrates in transfected cells. In this context, the mutant core RAG proteins support V(D)J recombination with reduced efficiency and with different levels and types of recombination products (9-15).Although not required for the biochemistry of V(D)J recombination, the noncore regions of RAG1 and RAG2 are conserved throughout evolution. For example, sequence conservation across the entire RAG2 protein from pufferfish to humans is Ϸ60%, with conservation of the noncore C-terminal region slightly higher than the core domain (16). Therefore, the noncore regions of RAG1 and RAG2 may serve important accessory and͞or regulatory functions in chromosomal V(D)J recombination. Consistent with this notion, studies of Abelson murine leukemia virus-transformed pre-B cells have suggested that the noncore regions of RAG1 are important for IgH locus D H -to-J H rearrangement (17), whereas the C terminus of RAG2 is more important for V H -to-DJ H rearrangement than for D H -to-J H rearrangement (18). The C terminus of RAG2 is predicted to fold into a plant homeodomain (PHD) (19,20), a motif found in a number of chromatin-associated proteins including some that have chromatin-modifying activities (21-24).To investigate potential in vivo function of the RAG2 noncore region in chromosomal V(D)J recombination and normal lymphocyte development, we have generated and characterized mice containing specific replacement of the full-length endogenous RAG2 gene with a gene encoding the mouse core...
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