DNA sequence specificity of APOBEC family deaminases

• Main Author: Beale, R. C. L.
• Published: University of Cambridge 2007
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596493

APOBEC family deaminases are capable of causing mutations by deaminating cytosine in DNA to uracil. This is exploited in diversification of the repertoire of antibodies by somatic hypermutation, and also in restricting the spread of retroviruses. APOBEC family induced mutations are not distributed entirely at random throughout the genes they deaminate; rather each APOBEC family member has its own preferred local sequence that will be preferentially targeted. Work presented in this thesis elucidates these preferred motifs for a number of different deaminases and investigates the structural basis of their specificity using viral and bacterial genetic assays. To determine the local sequence specificities of APOBEC proteins active in <i>E. coli</i>, a novel selection system was devised based on the conditional-lethal <i>sacB</i> gene. By varying the activity and orientation of promoters it was possible to target mutations to a chromosomally integrated <i>sacB</i> gene under certain conditions. Selecting for viable mutants generated mutation spectra for the AID, APOBEC1 and APOBEC3G deaminases. This enabled their preferred sequence motifs to be identified and correlated with particular mutation patterns found <i>in vivo.</i>