Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Abstract

d Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG ؊/؊ cells, uracil levels increase by 11-to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG ؊/؊ splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels. When B lymphocytes are activated through antigen presentation, they acquire hypermutations in the immunoglobulin (Ig) genes, facilitating affinity maturation of antibodies. An enzyme, activation-induced deaminase (AID), initiates these events by converting cytosines in DNA to uracil (1-4). The introduction of this rare base into DNA leads to a very high frequency of base substitution mutations in the Ig variable domain (known as somatic hypermutations [SHMs]; reviewed in references 5 and 6). Generation of uracils is also the first step in the creation of strand breaks in the switch regions of Ig genes, leading to the replacement of the constant domain with other domains such as ␥, in a process called class-switch recombination (CSR; reviewed in reference 7). AID also binds near the transcription start sites of a large number of actively transcribed genes (8) and mutates a number of additional genes, including those encoding BCL-6, MYC, PAX-5, and PIM1 (9-12). The uracils generated by AID are thought to be removed by the nuclear form of the uracil-DNA glycosylase, UNG2, creating abasic sites that are repaired by error-prone copying mechanisms causing hypermutations (13,14). Another uracil-DNA glycosylase, SMUG1, is normally considered the backup enzyme for UNG2 (15), but overproduction of SMUG1 is required for it to complement an UNG Ϫ/Ϫ mutant during CSR (16). Additionally, DNA mismatch repair (MMR) also plays a role in shaping the mutation spectrum in SHM (17).There is a strong connection between expression of AID and cancers in animals. Constitutive expression of AID in mice causes T cell cancers (18). Many human...

Functions and Malfunctions of Mammalian DNA-Cytosine Deaminases

“…In a different study, uracils in DNA were replaced with biotin-containing tags, and total genomic uracils were quantified following ex vivo stimulation of WT and UNG −/− splenocytes. 94 …”

APOBEC3A damages the cellular genome during DNA replication

“…56 In cells induced to express A3A, genomic uracil quantity in replicating cells was double that of resting cells (Fig. 3E).…”

“…56 Briefly, genomic DNA was extracted and treated with E. coli uracil DNA glycosylase (New England BioLabs) and an aldehyde-reactive probe (Dojindo Laboratories). The DNA samples were spotted onto a positively charged nylon membrane (Immobilon-Ny; Millipore) and membranes were then incubated in a solution containing streptavidin-Cy5 (GE Healthcare).…”

A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair

“…An advantage of the use of ARP in labeling AP sites is that multiple samples can be processed in parallel and the reaction products can be spotted on a membrane to create an ELISA-like assay. The biotin is subsequently bound with streptavidin that is linked with horseradish peroxidase (10) and incubated with chemiluminescent substrate, or directly bound to fluorescently tagged streptavidin (15) to obtain an optical readout. ARP has been used to determine AP sites in different mammalian tissues (16), to monitor changes in AP sites during aging (17) and AP sites generated as a result of treatment of cells with carcinogens (18).…”

“…It has also been adapted to quantify genomic uracils by excising uracils by uracil-DNA glycosylase to create AP sites followed by ARP treatment (19–21). It has been used to determine uracil levels in normal and repair-deficient Escherichia coli cells (20–22), in normal mammalian tissue (15,19) and in cancer cells (15). …”