Analysis of a Single-stranded DNA-scanning Process in Which Activation-induced Deoxycytidine Deaminase (AID) Deaminates C to U Haphazardly and Inefficiently to Ensure Mutational Diversity

Abstract

Enzymes that scan single-stranded (ss) DNA have been studied far less extensively than those that scan double-stranded (ds) DNA. Activation-induced deoxycytidine deaminase (AID) deaminates C to U on single-stranded DNA to initiate immunological diversity. Except for processive deaminations favoring WRC hot motifs (W ‫؍‬ (A/T) and R ‫؍‬ (G/C)), the rules governing AID scanning remain vague. Here, we examine the patterns of deaminations on naked single-stranded DNA and during transcription of dsDNA by embedding cassettes containing combinations of motifs within a lacZ mutational reporter gene. Deaminations arise randomly, spatially distributed as isolated events and in clusters. The deamination frequency depends on the motif and its surrounding sequence. We propose a random walk model that fits the data well, having a deamination probability of 1-7% per motif encounter. We suggest that inefficient, haphazard deamination produces antibody diversity associated with AID.An understanding of AID 2 scanning and C deamination is central to deciphering the many mysteries surrounding its essential role in initiating antibody diversity. AID deaminates C to U in B cells in variable (V) and switch (S) regions of immunoglobulin genes that are undergoing active transcription. Deaminations in the V region are responsible for initiating somatic hypermutation, and those in the S region initiate class switch recombination (1-3). These regulatory processes are multifaceted with poorly defined basic properties. How is AID targeted to the V and S regions, for example? What are its principal partnering proteins and regulatory elements? Why does AID fail to target concurrently transcribed non-V and non-S regions? Or if AID were to attack other transcribed regions, do these undergo efficient base excision and mismatch repair (1-3)? AID is also involved in restriction of retroviral transposition (4) and in editing of hepatitis B viral DNA (5). Recently, AID has taken on an entirely new "life," appearing to play a central role in active demethylation processes in stem cells, involving the deamination of 5-methylcytosine (6 -9). These recent developments and open questions about AID are discussed in several reviews (10 -13).We have previously shown that AID acts on ssDNA processively by catalyzing numerous C to U deaminations in trinucleotide target motifs on the same substrate molecule prior to acting on another DNA substrate (14, 15). WRC hot motifs (W ϭ (A/T) and R ϭ (A/G)) are favored targets for deamination over SYC cold motifs (S ϭ (G/C) and Y ϭ (T/C)) (14 -17). In light of the uncertainties of how AID functions in complex biological systems, as an initial step it is both essential and timely to study the behavior of AID in the simplest cases, when it acts on naked ssDNA and on actively transcribed dsDNA. It is timely because AID is the subject of numerous recent studies, and enzymes that scan ssDNA are poorly understood compared with those that scan dsDNA. It is essential because a fundamental knowledge of how AID scans DNA in the ab...

Overlapping hotspots in CDRs are critical sites for V region diversification

“…These studies confirmed that purified AID had a preference for the AGCT, even on single-stranded DNA. However, it is difficult to extrapolate the in vitro findings to the in vivo events because in the biochemical system, AID both is limiting and is processive through a slide and jump mechanism (56,57). The Ramos cells provide a good cellular model for the initial mutational events that occur in vivo because AID is constantly expressed and the relative susceptibility of the different AID hotspots to mutation is similar to that seen in mice (69).…”

“…Others have also seen differences between biochemical studies of the top (untranscribed) and bottom (transcribed) strands and attributed these to the fact that the in vivo bottom strand is heavily occupied by RNA polymerase and its many associated factors, including exosomes, so it is more difficult to duplicate the process biochemically (14,51). Because AID is limited and processive through a slide-and-jump mechanism in this biochemical assay (56,57), these results suggest that even on single-stranded DNA, the AGCT motifs were more likely to undergo AID-induced deamination and, through AID's processivity in this in vitro system, increase the frequency of mutation throughout the V region.…”

A Mathematical Model for Scanning and Catalysis on Single-stranded DNA, Illustrated with Activation-induced Deoxycytidine Deaminase

“…The most salient mathematical insight is that although the combined distribution for all of the clones with C3 U deaminations is used to decipher the displacement dynamics of AID on ssDNA, the catalysis does not appear explicitly in the expression for the two-point correlation function. In other words, although scanning and catalysis are closely coupled physically, scanning per se can be determined independently from catalysis using the mathematics, which we have shown is not the case for computer simulations of random scanning and catalysis by AID (25). This key result that enables scanning to be determined independently is contained in Equation 5 (see "Experimental Procedures").…”

“…1A). AID acts processively, catalyzing variable numbers of C3 U deaminations on the same ssDNA substrate (18,25). A "typical" DNA clone depicting AID displacement and catalysis is illustrated in Fig.…”

“…Previous biochemical studies show that AID binds to ssDNA, scans randomly (short hops/slides, longer jumps/intersegmental transfers), and catalyzes C deaminations, favoring WRC motifs (W ϭ A/T, R ϭ A/G) (18,25) (see Fig. 1A).…”