Analysis of coding joints formed by some of these mutants revealed excessive deletions, frequent use of short sequence homologies, and unusually long palindromic junctional inserts, known as P nucleotides, that result from aberrant hairpin opening. We found several amino acids outside of the canonical nonamer-binding domain that are critical for DNA binding, several step arrest mutants with defects in nicking or hairpin formation, and four RAG-1 mutants defective specifically for joining. To search for additional catalytic amino acids and to better define the functional anatomy of RAG-1, we mutated all 86 conserved basic amino acids to alanine and evaluated the mutant proteins for DNA binding, nicking, hairpin formation, and joining. RAG-1 contains three acidic active-site amino acids that are thought to coordinate catalytic metal ions. Although both RAG-1 and RAG-2 are required for all steps of V(D)J recombination, little is known about the specific contribution of either protein to these steps.
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