Project: Research project

Project Details


DESCRIPTION (Adapted from Investigator's abstract): The vertebrate
immune response is largely dependent on a vast population of antigen-
receptors. Much of the diversity in these molecules is now understood
to arise from a site-specific DNA recombination process called V(D)J
recombination. Inherited coding regions are cut and pasted in a reaction
that assembles mature genes. The combinatorial and mechanistic
contributions to the product create a degree of diversity that could not
be obtained by inheriting preformed genes. Two proteins, RAG1 and RAG2,
play an essential role in the reaction. In the past two years, these
two proteins have been shown to cut the target DNA at the appropriate
site in an in vitro reaction that contains no other factors. A detailed
study of the role of these two proteins alone and in concert with other
proteins in the later steps of the reaction is vital to an understanding
of this critical mechanism. The specific aims are: (1) Determine the
contacts between individual RAG proteins and DNA. A DNA binding assay
based on UV crosslinking will be used to identify the DNA binding
domains of the two proteins. The contact sites will be determined on
the DNA and on the protein. Definition of the DNA binding domain will
also allow better understanding of the architecture of the two proteins,
and delineation of domains that should be involved in protein-protein
contacts. (2) Demonstrate protein-protein interactions involving the
RAG proteins alone. Neither protein shows biochemical activity alone,
and the two proteins together are capable of cleaving the substrate DNA,
not only at single signals, but in a concerted manner at pairs of
signals. Protein-protein interactions are likely to be involved. (3)
Study protein and DNA interactions in the putative larger complex.
Mutant cells, defective in certain DNA repair proteins, are unable to
complete the V(D)J recombination reaction. The proteins recognized by
this behavior are potential participants in later steps in the
recombination reaction. Since one candidate protein for this complex
is a kinase, particular attention will be directed to phosphorylation
events that may accompany complex formation. This recombination
reaction is an essential step in the development of a normal immune
system. Furthermore, errors in this reaction have been implicated in
creating the chromosomal translocations that seem to precipitate many
childhood leukemias. Finally, this metabolic pathway is connected to
DNA break repair and cell cycle regulation which gives the study of
V(D)J recombination a broader significance.
Effective start/end date6/1/985/31/07


  • Cell Biology
  • Genetics
  • Medicine(all)
  • Immunology and Microbiology(all)


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