Class switch recombination is among the least understood types of genetic recombination. It is one of two types of DNA recombination in mammals important for the development of the immune system; yet, it sometimes occurs aberrantly, resulting in lymphoid malignancy. In fact, the c-myc gene translocates to the Ig class switch regions in all sporadic Burkitt's lymphomas (small, noncleaved cell lymphomas); in about 20 percent of diffuse, large cell non-Hodgkins lymphomas; and in a substantial fraction of HIV-associated non-Hodgkins lymphomas. It appears that transcription plays a key role in activating transcription for switch recombination. The efficiency and some of the targeting of class switch recombination can be recapitulated on minichromosomal substrates. These studies indicate that there is strand-specificity in the transcriptional targeting. Transcription in one direction results in recombination, whereas transcription in the opposite direction does not. This observation is more noteworthy in light of observations that transcription of switch regions in a cell-free system results in a stable RNA:DNA hybrid. Based on these cellular and cell-free studies indicating the importance of transcription and its orientation- specificity, we have devised a series of Aims directed at dissecting the nucleic acid and protein biochemistry of class switch recombination. First, a mouse model is proposed to test the orientation requirement of class switch recombination in the chromosome. Second, two mouse models are proposed to examine the joining step of switch recombination. One compares Ku knockout with DNA-PK mutants, and the other examines the DNA ligase IV knockout, based on our recent data indicating that it is the DNA ligase responsible for the large majority of DNA end joining in yeast and, probably, in V(D)J recombination. Third, we use the extrachromosomal assay system to study the length, targeting, and sequence requirements for class switch recombination. Fourth, we study the nucleic acid configuration of the non-B DNA configuration at switch regions in a cell-free system as the basis for further experiments in cellular systems. Fifth, we search for enzymatic activities in extracts that carry out the cleavage step in class switch recombination.
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