A single-stranded substrate was used in these assays to ensure that double-strand-specific glycosylases, including SMUG1, would not act around the uracil

A single-stranded substrate was used in these assays to ensure that double-strand-specific glycosylases, including SMUG1, would not act around the uracil. enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is usually 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 this uracil creation/excision balance is usually restored during establishment of cell lines, fixing the genomic uracil load at high levels. INTRODUCTION Rusalatide acetate 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,C4). The introduction of this rare base into DNA leads to a very high frequency of base substitution mutations in the Ig variable Rusalatide acetate domain (known as somatic hypermutations [SHMs]; reviewed in recommendations 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 Rusalatide acetate 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, Rabbit Polyclonal to ALPK1 and PIM1 (9,C12). 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?/? 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 B cell lymphomas and some leukemias that arise during the maturation of B lymphocytes in germinal centers (GC) constitutively express AID (19, 20). This is probably because AID is required for generating both of the double-strand breaks involved in the c-myc/IgH translocations that are a hallmark of B cell cancers (21, 22). Additionally, UNG?/? mice develop B cell hyperplasia and lymphomas at higher frequency than normal mice, suggesting that B cell maturation in the absence of UNG promotes oncogenic transformation (23). Based on such observations, it has been suggested that uracils generated by AID cause mutations and/or strand breaks that drive cellular transformation in some of the B cells undergoing maturation in germinal centers and resulting in hematopoietic cancers (24). Despite the wide acceptance of the idea that AID converts cytosines in DNA to uracil, no study has yet identified or quantified uracils in B cell tumor genomes. The only reports of uracils created by AID in normal B Rusalatide acetate cells have been in mouse models of antibody maturation that have focused only around the Ig genes (25, 26). Furthermore, the balance between uracil creation in the B cell genome by AID and its removal by UNG2 or other repair enzymes has not been examined. For example, it is not known whether the targeting of a large number of genes by AID (8, 27, 28) results in uracil accumulation in the genomes of B cells undergoing normal maturation in germinal centers. It is also not known whether the B cell cancers that constitutively express AID at high levels have enhanced repair capabilities that.