The activation-induced deoxycytidine deaminase (AID) is a member the Apobec family of enzymes that catalyzes C to U deamination on ssDNA tri-nucleotide motifs. In B cells, it is required to generate antibody diversity by initiating somatic hypermutation (SHM) in the variable region of immunoglobulin genes and class-switch recombination (CSR) in immunoglobulin switch regions. In turn, SHM and CSR are required to generate high-affinity antibodies that bind and neutralize invading antigens. Thus, AID plays an indispensable role in causing mutational diversity to enhance fitness and optimize the immune response. Here, we have used single-molecule fluorescence resonance energy transfer (smFRET) to visualize co-transcriptional scanning of AID. Our data show that AID can follow an active RNA polymerase directionally and processively with speeds upwards of 200 nt/s. However, transcription-stalling leads to bidirectional scanning in the transcription bubble, which in turn, provides AID the necessary time window to carry out deaminations. In bear ssDNA, AID scanning is slow (~1 s-1), random and bi-directional. The enzyme remains bound to the ssDNA for ~250 s on average. During this time, it can scan large (>70 nt) ssDNA regions, and it exhibits ‘quasi-localization’ near favorable deamination motifs. AID also creases the ssDNA during scanning in a sequence dependant manner.


Friday, October 18, 2013, 10:30. Seminar Room

Hosted by Prof. Melike Lakadamyali