The deletion looping out model of switch recombination predicts that the intervening DNA between S regions will be excised as a circle. Circular excision products of switch recombination have been isolated from mitogen activated splenic B cells. This model predicts that double strand breaks (DSBs) are formed in switch regions as an intermediate in the recombination reaction. We have found sequence specific, blunt ended DSBs in Sgamma3 DNA. These breaks are limited to B cells and confined to the switch region. We have also studied a Ku80-/- mouse for which the B cell compartment was reconstituted by knock-in of the IgH and L chain genes. This analysis indicates that Ku80 is required in the switch recombination reaction. The presence of DSBs in S DNA and the requirement for Ku strongly suggests that switch recombination precedes through a DNA end joining mechanism. We have made significant progress toward establishing an extrachromosomal switch substrate assay for isotype switching. Using this assay we provide evidence for class specific factors involved in the recombination process. Based on these findings, I propose to further investigate the specificity of factors involved in switch recombination by construction of additional switch plasmids with defined isotypes. These plasmids will be analyzed in normal B cells and cell lines with defined capacity for switch recombination. The function of Ku80 will be further analyzed using the switch plasmids. The role of transcription in cleavage of S regions during recombination will be explored. Finally, a major effort toward cloning factors involved in the switch recombination reaction is proposed.
