Class switch DNA recombinations are important for isotype switching in B cells and also appear to be involved in chromosomal translocations of some oncogenes. However, the mechanisms of switching and the processes that target these mechanisms to certain DNA regions are still not known. Switch (S) regions containing tandemly repeated sequences are located upstream of all H-chain antibody constant region genes and are the target of the switch recombinational machinery. However, the roles of S regions in targeting are unclear. We have found that the S? tandem repeat region is not required for switching but does play a role in providing highly efficient switching. In mice lacking the DNA mismatch repair protein, Msh2, we have also found that the S? tandem repeats are critical for switching, indicating that different DNA regions need different proteins to complete switch recombination. Finally, measurements of switch site distributions in mice that lack either the S? tandem repeats or the Msh2 protein show shifts in switch targeting. These shifts indicate that a 4-5 kb domain downstream of the l? promoter is accessible for switching even if the S? tandem repeats are not within this domain. In addition, in the absence of Msh2, switching is focused to the tandem repeat region within the domain. The current proposal seeks to further analyze the targeting of switching by S regions using a variety of mutant mouse strains that affect the isotype switching process. First, the roles of S? region sequences in targeting switch recombination through possible formation of R-loop structures, or by promoting specific chromatin structures will be analyzed in wild-type and mutant mice. Second, the roles of the Mlh1 and Exo1 mismatch repair proteins in switching will be compared to the role of Msh2 to determine whether these proteins affect the same or different pathways in the switching mechanism. Third, the abilities of S? and l? regions in regulating switch targeting will be analyzed by relocating these sequences and assessing whether switch targeting is also relocated. Finally, we will assess the importance of the AID protein, which is critical in initiating switch recombination, for the u transgene chromosomal translocations that were discovered in our laboratory. Although aberrant targeting of oncogenes by the switch mechanism has been suggested to be involved in some oncogene:lgH translocations, recent studies by another laboratory have indicated that these translocations do not involve AID. If ? transgene translocations also do not involve AID then this would provide a convenient model system for genetic analyses of the sequences and proteins important for the IgH translocation process.
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