ABSTRACT
RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination
signal sequences through sequential nicking and transesterification
reactions to yield blunt signal ends and coding ends terminating in a
DNA hairpin structure. Ubiquitous DNA repair factors then mediate the
rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule,
which limits rearrangement to signal sequences bearing different
lengths of DNA (12 or 23 base pairs) between the conserved heptamer and
nonamer sequences to which the RAG proteins bind. Both RAG proteins
have been subjected to extensive mutagenesis, revealing residues
required for one or both cleavage steps or involved in the DNA
end-joining process. Gain-of-function RAG mutants remain unidentified.
Here, we report a novel RAG-1 mutation, E649A, that supports elevated
cleavage activity in vitro by preferentially enhancing hairpin
formation. DNA binding activity and the catalysis of other DNA strand
transfer reactions, such as transposition, are not substantially
affected by the RAG-1 mutation. However, 12/23-regulated synapsis does
not strongly stimulate the cleavage activity of a RAG complex
containing E649A RAG-1, unlike its wild-type counterpart.
Interestingly, wild-type and E649A RAG-1 support similar levels of
cleavage and recombination of plasmid substrates containing a 12/23
pair of signal sequences in cell culture; however, E649A RAG-1 supports
about threefold more cleavage and recombination than wild-type RAG-1 on
12/12 plasmid substrates. These data suggest that the E649A RAG-1
mutation may interfere with the RAG proteins' ability to sense
12/23-regulated
synapsis.