Abstract
During V(D)J recombination, RAG targeting to correct sites versus off-target sites relies on both DNA sequence features and on chromatinmarks. Kinetic analysis using the rst highly active full-length puried RAG1/RAG2 complexes has now allowed us to dene the important catalytic features of this complex.We found that the overall rate of nicking, but not hairpinning, is critical for the discrimination between correct (optimal) versus off-target (suboptimal) sites used in human T-cell lymphomas, and we show that the C-terminal portion of RAG2 is required for this. This type of kinetic analysis permits us to analyze only the catalyt-ically active RAG complex, in contrast to all othermethods, which are unavoidably confounded bymixture with inactive RAG complexes.Moreover, we can distinguish the twomajor features of any enzymatic catalysis: the binding constant (K D) and the catalytic turnover rate, k cat. Beyond aminimal essential threshold of heptamer quality, further suboptimal heptamer deviations primarily reduce the catalytic rate constant k cat for nicking. Suboptimal nonamers reduce not only the binding of the RAG com-plex to the recombination site (K D) but also the catalytic rate constant, consistent with a tight interaction between the RAG com-plex and substrate during catalysis. These features explainmany aspects of RAG physiology and pathophysiology.