ESSENTIAL RESIDUES IN V(D)J RECOMBINATION SIGNALS

Recombination signal sequences for V(D)J joining consist of a conserve d heptamer (CACAGTG) and a nonamer (ACAAAAACC) separated by a spacer o f a constant length (12 bp or 23 bp). In the present study, we have an alyzed various recombination signal mutations for their effects in V(D )J joining. Using a retroviral vector, we introduced mutant substrates stably into pre-B cells, and assayed recombination using the lacZ gen e as a reporter. This method allowed us to study recombination in a si ngle copy within the context of the host cell chromosome. Because this assay did not show any detectable background, it was quite useful in the analysis of low level recombinations. In the heptamer, mutations i n the first three residues severely dropped the joining rates. Among t hem, the first residue adjacent to the recombination site was found to be most essential. Although mutations in the heptamer reduced the joi ning rate to various extents, they did not lower the site-specificity of recombination. With regard to the nonamer, the presence of three co nsecutive A residues was necessary for efficient recombination. Furthe rmore, the nucleotides flanking the A-rich core needed to be other tha n A residues, probably marking the border of the A-stretch. This may b e important when the recombinase measures the distance between the hep tamer and the nonamer to satisfy the 12/23-bp spacer rule.