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Departments of
* Immunology and
Biochemistry, University of Washington School of Medicine, Seattle, WA 98195
Rearranged Ig V regions undergo activation-induced cytidine deaminase (AID)-initiated diversification in sequence to produce either nontemplated or templated mutations, in the related pathways of somatic hypermutation and gene conversion. In chicken DT40 B cells, gene conversion normally predominates, producing mutations templated by adjacent pseudo-V regions, but impairment of gene conversion switches mutagenesis to a nontemplated pathway. We recently showed that the activator, E2A, functions in cis to promote diversification, and that G1 phase of cell cycle is the critical window for E2A action. By single-cell imaging of stable AID-yellow fluorescent protein transfectants, we now demonstrate that AID-yellow fluorescent protein can stably localize to the nucleus in G1 phase, but undergoes ubiquitin-dependent proteolysis later in cell cycle. By imaging of DT40 polymerized lactose operator-
R cells, in which polymerized lactose operator tags the rearranged R gene, we show that both the repair polymerase Pol
and the multifunctional factor MRE11/RAD50/NBS1 localize to R, and that R/Pol colocalizations occur predominately in G1 phase, when they reflect repair of AID-initiated damage. We find no evidence of induction of 
-H2AX, the phosphorylated variant histone that is a marker of double-strand breaks, and Ig gene conversion may therefore proceed by a pathway involving templated repair at DNA nicks rather than double-strand breaks. These results lead to a model in which Ig gene conversion initiates and is completed or nearly completed in G1 phase. AID deaminates ssDNA, and restriction of mutagenesis to G1 phase would contribute to protecting the genome from off-target attack by AID when DNA replication occurs in S phase.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grants R01 GM39799 and R01 GM41712 (to N.M.) and National Institutes of Health Predoctoral Training Programs T32 GM07223 and T32 AG00057 (to E.C.O.).
2 E.C.O. and M.Y. contributed equally to this work.
3 Current address: Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720.
4 Address correspondence and reprint requests to Dr. Nancy Maizels, Department of Immunology, 474A HSB, University of Washington School of Medicine, Seattle, WA 98195-7650. E-mail address: maizels{at}u.washington.edu
5 Abbreviations used in this paper: AID, activation-induced cytidine deaminase; AP, abasic; DAPI, 4,6-diamidino-2-phenylindole; DSB, double-strand break; IR, ionizing radiation; LacI, lactose repressor; LMB, leptomycin B; MRN, MRE11/RAD50/NBS1; NES, nuclear export signal; NHEJ, nonhomologous end joining; PolyLacO, polymerized lactose operator; RFP, red fluorescent protein; sIgM, surface IgM; Ugi, uracil-DNA glycosylase inhibitor; UNG, uracil-DNA glycosylase; YFP, yellow fluorescent protein.
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