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Q M Eastman

Publications and source records attributed to Q M Eastman.

7 recordsLinked to original sources
PubMed1999

Detection of RAG protein-V(D)J recombination signal interactions near the site of DNA cleavage by UV cross-linking.

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.

Animals↗
PubMed1998

Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system.

Immunoglobulin and T-cell-receptor genes are assembled from component gene segments in developing lymphocytes by a site-specific recombination reaction, V(D)J recombination. The proteins encoded by the recombination-activating genes, RAG1 and RAG2, are essential in this reaction, mediating sequence-specific DNA recognition of well-defined recombination signals and DNA cleavage next to these signals. Here we show that RAG1 and RAG2 together form a transposase capable of excising a piece of DNA containing recombination signals from a donor site and inserting it into a target DNA molecule. The products formed contain a short duplication of target DNA immediately flanking the transposed fragment, a structure like that created by retroviral integration and all known transposition reactions. The results support the theory that RAG1 and RAG2 were once components of a transposable element, and that the split nature of immunoglobulin and T-cell-receptor genes derives from germline insertion of this element into an ancestral receptor gene soon after the evolutionary divergence of jawed and jawless vertebrates.

Amino Acid Sequence↗
PubMed1997

Nicking is asynchronous and stimulated by synapsis in 12/23 rule-regulated V(D)J cleavage.

The first step in DNA cleavage at V(D)J recombination signals by RAG1 and RAG2 is creation of a nick at the heptamer/coding flank border. Under proper conditions in vitro the second step, hairpin formation, requires two signals with spacers of 12 and 23 bp, a restriction referred to as the 12/23 rule. Under these conditions hairpin formation occurs at the two signals at or near the same time. In contrast, we find that under the same conditions nicking occurs at isolated signals and hence is not subject to the 12/23 rule. With two signals the nicking events are not concerted and the signal with a 12 bp spacer is usually nicked first. However, the extent and rate of nicking at a given signal are diminished by mutations of the other signal. The appearance of DNA nicked at both signals is stimulated by more than an order of magnitude by the ability of the signals to synapse, indicating that synapsis accelerates nicking and often precedes it. These observations allow formulation of a more complete model of catalysis of DNA cleavage and how the 12/23 rule is enforced.

DNA↗
PubMed1997

Coding joint formation in a cell-free V(D)J recombination system.

V(D)J recombination assembles the variable portion of antigen receptor genes in developing lymphocytes and is the only site-specific recombination reaction known in vertebrates. A cell-free system has been established that performs DNA cleavage, end processing, and joining to yield V(D)J coding joints that exhibit structural features similar to those formed in vivo. The reaction has the expected substrate, metal ion, and RAG protein requirements. The efficiency of coding joint formation is reduced dramatically by uncoupling the cleavage and joining portions of the reaction, indicating that a postcleavage coding end complex facilitates joining. By varying the reaction conditions, nucleotide loss from coding ends and heterogeneity of coding joints can be regulated. This cell-free system provides a novel tool for detailed mechanistic analyses of the end processing and joining steps of V(D)J recombination.

Cell-Free System↗
PubMed1996

RAG1 mediates signal sequence recognition and recruitment of RAG2 in V(D)J recombination.

Recent studies have demonstrated that DNA cleavage during V(D)J recombination is mediated by the RAG1 and RAG2 proteins. These proteins must therefore bind to the recombination signals, but the specific binding interaction has been difficult to study in vitro. Here, we use an in vivo one-hybrid DNA binding assay to demonstrate that RAG1, in the absence of RAG2, can mediate signal recognition via the nonamer, with the heptamer acting to enhance its binding. A region of RAG1 with sequence similarity to bacterial invertases is essential for DNA binding. Localization of RAG2 to the signal is dependent upon the presence of RAG1 and is substantially more efficient with a 12 bp spacer signal than with a 23 bp spacer signal.

Amino Acid Sequence↗
PubMed1996

Initiation of V(D)J recombination in vitro obeying the 12/23 rule.

V(D)J recombination, the process that assembles antigen-receptor genes, is directed by signal sequences flanking the DNA segments to be joined. Signals consist of a conserved heptamer and nonamer separated by a spacer of either 12 or 23 base pairs. Recombination occurs almost exclusively between two signals with spacers of different lengths. This restriction, called the '12/23 rule', governs the organization and pattern of rearrangement of antigen-receptor loci. In vitro work demonstrating the direct roles of the Rag proteins in the initiation of V(D)J recombination did not recreate the 12/23 rule. Instead, double-strand breaks were formed efficiently at isolated signals. Here we show that extracts made from a lymphoid cell line that expresses truncated forms of the Rag1 and Rag2 proteins have a signal-cutting activity that obeys the 12/23 rule. Cleavage at the two signals is concerted and requires their synapsis, and mutations of one signal prevent cleavage at both.

Base Sequence↗