NMR-Distance Geometry Studies of Helical Errors and Sequence Dependent Conformations of DNA in Solution

Patel, Dinshaw J.; Shapiro, Lawrence; Hare, Dennis R.

doi:10.1007/978-1-4612-3800-3_8

Cited by 7 publications

(3 citation statements)

References 86 publications

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“…Second, the RAG proteins can specifically cleave an RSS which is entirely singlestranded, suggesting that some unwinding might naturally occur during cleavage (5,24). Third, the CACA sequence of the heptamer is likely to have an unusual structure in which the DNA is partially unwound (4,23,35). Thus, it remains likely that access to RSS and therefore V(D)J recombination may be regulated by the state of DNA unwinding or the potential for local distortion.…”

Section: Discussionmentioning

confidence: 99%

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

Akamatsu

Oettinger

1998

Molecular and Cellular Biology

124

153

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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...

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Section: Discussionmentioning

confidence: 99%

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

Akamatsu

Oettinger

1998

Molecular and Cellular Biology

124

153

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“…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

Molecular and Cellular Biology

124

140

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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 ...

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“…Cold Spring Harbor Laboratory Press on April 3, 2019 -Published by genesdev.cshlp.org Downloaded from cleotide in the context of a larger oligonucleotide displays partial base unstacking, lower melting temperature, base-pair tilt, and displacement from the DNA helical axis (Cheung et al 1984;Patel et al 1988}. These unique structural features may be important for recognition or cleavage by the V(D)J recombinase.…”

Section: Genes and Development 1465mentioning

confidence: 99%

Coding end sequence can markedly affect the initiation of V(D)J recombination.

Gerstein¹,

Lieber²

1993

Genes & Development

111

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(Fig. 1 ). V(D)J recombination occurs between two gene elements: one with a 12-bp spacer between the heptamer and nonamer (12-signal), and the other with a 23-bp spacer between them (23-signal). The sequence directly adjacent to the heptamer, which ultimately forms the recombinant junction within the antigen receptor variable exon, is referred to as the coding end. Coding end sequence has been assumed to be neutral with respect to the efficiency of recombination because (1) the several hundred antigen receptor-coding el-'Corresponding author. ement sequences are diverse, and (2) signal sequences, with no additional immunoglobulin or TCR locus sequence, are necessary and sufficient to direct V(D)J recombination of exogenous substrates Hesse et al. 1987Hesse et al. , 1989. Here, we report a systematic comparison of the contribution to recombination efficiency of coding end sequences. Contrary to expectation, we find that nucleotide sequence at the coding end can have a large impact on the efficiency of V(D)J recombination. This finding has both mechanistic and developmental relevance for generation of the antigen receptor repertoire. Results Experimental strategyWe evaluated the contribution of coding end sequence to V(D)J recombination efficiency, as measured by extrachromosomal recombination substrates Lieber et al. 1987). These substrates allow for a quantitative assessment of the fraction of plasmid molecules that have entered the cell and undergone V(D)J recombination. The substrates used in this study were constructed from the same parental plasmid, contain identical consensus recombination signals (Hesse et al. 1989), and vary only at the terminal 1-8

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NMR-Distance Geometry Studies of Helical Errors and Sequence Dependent Conformations of DNA in Solution

Cited by 7 publications

References 86 publications

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

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

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

Coding end sequence can markedly affect the initiation of V(D)J recombination.

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