Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase

Abstract

A functional immune system depends on the production of a wide range of immunoglobulin molecules. Immunoglobulin variable region (IgV) genes are diversified after gene rearrangement by hypermutation. In the DNA deamination model, we have proposed that deamination of dC residues to dU by activation-induced deaminase (AID) triggers this diversification. In hypermutating chicken DT40 B cells, most IgV mutations are dC --> dG/dA or dG --> dC/dT transversions, which are proposed to result from replication over sites of base loss produced by the excision activity of uracil-DNA glycosylase. Blocking the activity of uracil-DNA glycosylase should instead lead to replication over the dU lesion, resulting in dC --> dT (and dG --> dA) transitions. Here we show that expression in DT40 cells of a bacteriophage-encoded protein that inhibits uracil-DNA glycosylase shifts the pattern of IgV gene mutations from transversion dominance to transition dominance. This is good evidence that antibody diversification involves dC --> dU deamination within the immunoglobulin locus itself.

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID ‐accessible single‐stranded DNA

“…Either there was less deamination of the IGVL R target, or the resulting abasic sites were less likely to be processed through a mutagenic pathway (Fig 2A). To discriminate between these two possibilities, we inhibited the conversion of dU to abasic sites by expression of a bacteriophage‐derived uracil glycosylase (UNG) inhibitor (UGI) (Di Noia & Neuberger, 2002). Inhibition of UNG leads to direct replication of the unexcised uracils resulting exclusively in C‐to‐T transitions, effectively revealing the “footprint” of deamination by AID (Xue et al , 2006).…”

“…Wild‐type and h3.3 cells overexpressing hAIDup were transfected with the UGI‐expression vector pEF‐UGI (Di Noia & Neuberger, 2002). Transfectants were selected in medium containing puromycin, and individual clones picked and maintained in 24‐well plates for 36 cell divisions.…”

Immunoglobulin gene conversion in chicken DT40 cells largely proceeds through an abasic site intermediate generated by excision of the uracil produced by AID‐mediated deoxycytidine deamination

“…Enzyme activity in Ugi transfectants was monitored using 5 ? g of undiluted cell extract in a 1.5-h incubation at 37°C with a double-stranded oligonucleotide containing a single centrally located dU as substrate [6]. were established that expressed UDG-inhibitor protein (Ugi) encoded by the bacteriophage PBS2 [9,10].…”

“…Mechanistically, both processes appear to be triggered by activation-induced deaminase (AID)-catalyzed deamination of deoxycytidine (dC) to deoxyuridine (dU) within the IgV gene [1][2][3][4][5][6]. Although gene conversion is presumably achieved by recombination-mediated repair of the resultant DNA lesion using proximal IgV pseudogenes [7], the precise mechanism remains obscure.…”

Forced expression of AID facilitates the isolation of class switch variants from hybridoma cells

“…These changes in isotype can be very important, since various isotypes are distributed differently in the body, have different half-lives in the circulation and carry out distinct subsets of effector functions (Stavnezer, 2000). Both SHM and CSR require activation induced cytidine deaminase (AID), which converts deoxycytidines in the V and switch regions to uracil and initiates both processes (Muramatsu et al, 1999;Di Noia et al, 2002;Manis et al, 2002;Bransteitter et al, 2003). Patients that are genetically deficient in AID make only low affinity IgM antibodies and die of infections if they are not treated with hyperimmune immunoglobulins (Quartier et al, 2004).…”