Antibody maturation occurs in response to invasion of a foreign agent such as a virus and creates antibody proteins that have greater affinity towards the invading antigens. Activation-induced cytidine deaminase (AID) is required for this process and its essential role in the fight against infections is evidenced by the fact that individuals that are unable to make active AID suffer from hyper-IgM syndrome. An important feature of the role of AID in promoting somatic hypermutations (SHM) and class switch recombination -2 processes essential for antibody maturation - is the requirement for the transcription of the immunoglobulin gene. We propose here to investigate the transcription-dependence of SHM. We outline here experiments in vitro, in Escherichia coli and in mammalian cells that will investigate the role of AID in promoting cytosine to thymine (C to T) and other base substitutions. Specifically, we will investigate the effects of supercoiling, R-loop formation and other structural features on strand bias and clustering of mutations. The experiments will use genetic assays in which kanamycin-resistant and phleomycin-resistant revertants respectively score C to T and non-C-to-T mutations. We will also develop a mammalian genetic system to analyze these 2 classes of mutations and study their strand bias during AID promoted hypermutations. Further, this system will be used to study the roles played by various DNA repair enzymes, DNA polymerases and accessory proteins in shaping the strand bias and spectrum of mutations responsible for antibody maturation.
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