How is the abasic site processed to create the single-strand breaks characteristic of hypermutating V regions [17]? How is the normally faithful uracil-DNA repair pathway diverted to have a recombinogenic or mutagenic outcome? How are the S regions juxtaposed for recombination? How do other highly expressed genes avoid mutation? We can look forward to progress in these and related areas in the near future. Acknowledgements We gratefully acknowledge support from the US National Institutes of Health, grant numbers R01 GM39799 and R01 GM41712 (N.M.) and F32 GM67482 (E.D.L.).. 1 Transcription-coupled mutagenesis initiates class-switch recombination and somatic hypermutation. The murine Salermide immunoglobulin heavy chain locus is shown (top line) with a rearranged variable (VDJ) region juxtaposed to the C constant region. AID is depicted traveling with the transcription apparatus (tailed arrows) at the expressed variable region and the activated S and S1 switch regions. The shaded box illustrates how AID first deaminates C KIAA1704 to U, and then uracil-DNA glycosylase removes U, leaving an abasic site. Subsequent steps generate single-strand breaks [17], which become substrates for mutagenic repair or recombination. Somatic hypermutation alters variable region sequence, and switch recombination joins a new constant region (C1) to the expressed variable region, producing an extrachromosomal DNA circle (bottom), which contains the deleted region. The final result is a heavy chain locus containing a mutated variable region (mutations are indicated by stars) and a chromosomal S/S1 junction (bottom). The first evidence that switch recombination and somatic hypermutation share any mechanistic components Salermide came in 2000, when a pair of papers [1,2] from Honjo, Durandy and collaborators showed that a single polypeptide, activation-induced deaminase (AID), induces both switch recombination and somatic hypermutation in mice [1] and humans [2]. AID is homologous to APOBEC1, a deaminase that edits a specific cytidine in the apolipoprotein B transcript to produce a nonsense codon that results in the expression of a truncated polypeptide [3]. This evolutionary relationship cast a long shadow over initial scenarios for how AID might function. It was anticipated that a specific RNA target for AID would soon be identified, and would in all likelihood be found to encode a master regulator in the form of a critical nuclease or transcription factor [4]. If such a target does exist, it has yet to be discovered. Instead, overwhelming experimental evidence supports the view that AID deaminates a C to a U in transcribed DNA. Transcription-coupled mutagenesis Evidence that AID acts in concert with transcription comes from a lovely recent paper [5]. The process of switch recombination is activated and targeted by the transcription of switch (S) regions, guanine-rich, 2-10 kilobase stretches of DNA located just upstream of those constant regions that participate in switch recombination (Figure ?(Figure1).1). Transcription of each S region is driven by a dedicated promoter that is responsive to specific transcription factors, which in turn are regulated by extracellular signals delivered by cytokines. Shimizu and collaborators [5] used chromatin immunoprecipitation to show that AID is present at the transcribed S1 and S switch regions in murine splenic B cells cultured in conditions that induce switching to 1 1 and (for immunoglobulins IgG1 and IgE, respectively); and, conversely, that AID could be found only at S1 and not at S if cells were cultured in conditions that stimulated switching to 1 1 but suppressed switching to . Furthermore, they obtained evidence for direct association between AID and RNA polymerase II. Thus, AID associates with the transcription apparatus to attack the transcribed S-region DNA (Figure ?(Figure11). Other data support and complement this conclusion. In experiments that draw on our understanding of how uracil in DNA is normally repaired, genetic analysis has produced compelling evidence that AID deaminates DNA. C to U deamination is common (100 C to U deamination events occur each day in each mammalian cell). The highly conserved pathways that repair uracil in DNA rely on uracil-DNA glycosylases to remove the uracil base, and apurinic endonucleases to nick the abasic site so Salermide that new DNA synthesis can recreate the duplex [6]. Reasoning that if AID deaminates DNA, repair of AID-induced lesions would depend upon uracil-DNA glycosylase activities, Neuberger and collaborators [7] showed that expression of AID in em Escherichia coli /em stimulates mutation at G?C base pairs, and that mutation levels are amplified in a strain deficient in uracil-DNA glycosylase. Climbing the evolutionary ladder, this same group showed that inhibition of uracil-DNA glycosylase alters the mutation Salermide spectrum in a hypermutating chicken bursal lymphoma cell line [8], and that switch recombination is impaired and the spectrum of hypermutation altered in mice deficient in uracil-DNA glycosylase [9]. Quite recently, Durandy and collaborators [10] reported a similar phenotype in humans deficient.