The deletion looping out model for Ig gene switch recombination predicts that the intervening DNA between H-chain S regions will be excised as a circle. Circular excision products of switch recombination have been isolated from mitogen activated B cells. The deletion looping out model also predicts that double strand breaks (DSBs) are formed within switch regions as an intermediate in the recombination reaction. In preliminary studies the Principal Investigator has detected sequence specific, 5'-phosphorylated, blunt ended DSBs in the Sgamma3 switch regions and has found evidence that these DSBs within in the Sgamma3 region are induced in LPS activated splenic B cells. The breaks are limited to B cells and apparently confined to the switch region. Previously, the investigator has defined the SNIP and SNAP DNA-binding proteins which bind two motifs in Sgamma3 DNA. More recent work indicates that the DSBs observed in Sgamma3 DNA are coincident with SNAP binding sites. Furthermore, a strong clustering of Sgamma recombination breakpoints within the SNIP and SNAP recognition sites in Sgamma DNA is observed. These findings suggest that switch recombination is an orderly process dependent on specific proteins and DNA motifs. The investigator plans to extend these findings in several ways. First, Sgamma3 specific DSBs will be analyzed for the presence of staggered ends. It is suggested that the presence of staggered ends might explain the observation of clustered recombination breakpoints. Second, the relationship or dependence of germline transcript expression and the induction of DSBs in normal B cells will be investigated. Third, the study of mitogen inducible DSBs will be extended to Smu and Salpha DNA. Finally, an extrachromosomal switch substrate assay is being developed. It is hoped that the ability to manipulate switch sequences and cis-flanking sequences on the switch substrate plasmid may allow a fine genetic analysis of features important to the recombination process. The study of switch recombination is likely to have implications for our basic understanding of genome stability. Aberrant switch recombination is responsible for a subset of B cell leukemias and lymphomas and has direct relevance to cancer.
| Wuerffel, R A; Ma, L; Kenter, A L (2001) NF-kappa B p50-dependent in vivo footprints at Ig S gamma 3 DNA are correlated with mu-->gamma 3 switch recombination. J Immunol 166:4552-9 |
| Shanmugam, A; Shi, M J; Yauch, L et al. (2000) Evidence for class-specific factors in immunoglobulin isotype switching. J Exp Med 191:1365-80 |
| Stavnezer, J; Bradley, S P; Rousseau, N et al. (1999) Switch recombination in a transfected plasmid occurs preferentially in a B cell line that undergoes switch recombination of its chromosomal Ig heavy chain genes. J Immunol 163:2028-40 |
| Kenter, A; Wuerffel, R (1999) Immunoglobulin switch recombination may occur by a DNA end-joining mechanism. Ann N Y Acad Sci 870:206-17 |
