Correlation of lac operator DNA imino proton exchange kinetics with its function.

124

153

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...

Section: Discussionmentioning

confidence: 99%

Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences

Akamatsu

Oettinger

1998

124

153

“…In addition, the purine-pyrimidine alteration of the consensus heptamer might help to stabilize altered DNA structures that have been observed at CACA stretches (48). Indeed, a large number of biophysical and biochemical experiments have established that (CA) n sequences such as the heptamer element are more bendable and less thermally stable than other DNA sequences (6,9,12) and that they are capable of adopting a variety of conformations (40,47,48). This malleability may be an important part of heptamer recognition by RAG proteins (2, 46) because of the considerable distortion of the DNA backbone necessary for hairpin formation.…”

Section: Discussionmentioning

confidence: 99%

Detection of RAG Protein-V(D)J Recombination Signal Interactions Near the Site of DNA Cleavage by UV Cross-Linking

Eastman

Villey

Schatz

1999

V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence-and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site organization of the RAG1-RAG2-RSS complex.The variable portions of antigen receptor genes are assembled from component gene segments during lymphocyte development by a process known as V(D)J recombination. The gene segments that undergo rearrangement are flanked by recombination signal sequences (RSSs), consisting of conserved heptamer (CACAGTG) and nonamer (ACAAAAA CC) elements separated by a spacer of either 12 or 23 bp. Recombination brings two protein-coding regions together in an imprecise junction and joins the two RSSs with the heptamer elements fused head-to-head. Recombination occurs predominantly between gene segments flanked by RSSs with spacers of unequal lengths. This "12/23 rule" helps to restrict the reaction to developmentally useful combinations (19).The products of the recombination activating genes, RAG1 and RAG2, are necessary for the initiation of V(D)J recombination (28, 41) and together catalyze the creation of a doublestrand break at the border of an RSS (23, 50). They first introduce a nick 5Ј to the first C nucleotide of the heptamer element. This exposes a 3Ј hydroxyl which then attacks the opposite strand of the DNA, creating a 5Ј-phosphorylated blunt end on the signal side and a closed hairpin on the side that will form protein-coding sequence (see Fig. 1A).The sequence requirements for hairpin formation are more stringent than those for initial nicking. In cells and in crude extracts, hairpin formation requires the formation of a synaptic complex involving the two RSSs, while nicking can occur at an isolated signal (10,11,13,44,45). The RAG proteins intrinsically prefer to cleave a 12/23 pair of signals (52); however, the ubiquitous DNA-bending proteins HMG-1 and HMG-2 strengthen this preference by boosting cleavage at the RSS with a 23-bp spacer and by aiding in the formation of a synaptic complex (39, 49). In addition, in a single-signal context, some mutations of the heptamer element substantially inhibit hairpin formation without preventing initial nicking (7, 31).Mutants of RAG1 have been identified a...

“…This finding again suggests that particular DNA structural features are important for RAG function. In addition, structural studies have shown that the CACA sequence has an intrinsically abnormal structure in which normal base pairing is disrupted (4,17,28). This abnormal structure may facilitate further the DNA distortion (see below) that is likely to be critical for the reaction; the formation of a perfect hairpin requires that the DNA at the tip of the hairpin be unpaired, and the intramolecular transesterification would require a kink on the bottom strand at the signal-coding border.…”

Section: Discussionmentioning

confidence: 99%

DNA Sequence and Structure Requirements for Cleavage of V(D)J Recombination Signal Sequences

Cuomo

Mundy

Oettinger

1996

124

140

Purified RAG1 and RAG2 proteins can cleave DNA at V(D)J recombination signals. In dissecting the DNA sequence and structural requirements for cleavage, we find that the heptamer and nonamer motifs of the recombination signal sequence can independently direct both steps of the cleavage reaction. Proper helical spacing between these two elements greatly enhances the efficiency of cleavage, whereas improper spacing can lead to interference between the two elements. The signal sequences are surprisingly tolerant of structural variation and function efficiently when nicks, gaps, and mismatched bases are introduced or even when the signal sequence is completely single stranded. Sequence alterations that facilitate unpairing of the bases at the signal/coding border activate the cleavage reaction, suggesting that DNA distortion is critical for V(D)J recombination.Immunoglobulin and T-cell receptor genes are assembled from gene segments by a series of site-specific recombination reactions collectively termed V(D)J recombination. By joining different combinations of V, D, and J coding segments, this process generates much of the enormous diversity in antigen receptor specificity that is required for a functional immune system (for recent reviews, see references 7 and 12). Expression of the RAG1 and RAG2 genes is required for this joining reaction to occur; lymphoid cells with targeted disruptions of either gene do not carry out V(D)J joining (15, 25). Conversely, expression of the RAG genes in fibroblasts is sufficient to allow recombination to occur in these nonlymphoid cells (16,23). Recent work has shown that the two RAG proteins together are the only protein factors required to carry out the initial cleavage step of V(D)J recombination in vitro (13).Recombination signal sequences (RSSs) flank all recombinationally active gene segments and are recognized by one or both RAG proteins in vitro (13). RSSs consist of conserved heptamer and nonamer motifs separated by a nonconserved spacer region of either 12 or 23 bp (called 12-spacer or 23-spacer RSSs). V(D)J recombination requires two RSSs, one of each spacer length. Because the segments at a given locus are flanked by the same arrangement of RSSs (e.g., 12-spacer signals at V and 23-spacer signals at J), this 12/23 rule restricts segment joining to events that could be biologically productive. V(D)J recombination results in the formation of a signal joint by the precise fusion of signal sequences heptamer to heptamer and a more varied coding junction at which base loss and addition have usually occurred.Mutational analysis of RSSs has shown that the consensus heptamer (5ЈCACAGTG3Ј) and nonamer (5ЈACAAAAACC 3Ј) sequences are also the optimal sequences for recombination of a plasmid substrate (9, 18). Indeed, the three bases of the heptamer (5ЈCAC3Ј) which are the most highly conserved are also found to have the strongest influence on recombination efficiency. A heptamer is required in each RSS for V(D)J recombination to occur; no recombinants are detected with substrates ...