RESUMO
Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4(-/-) cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Ligases/metabolismo , Switching de Imunoglobulina/genética , Recombinação Genética , Animais , Linhagem Celular , Núcleo Celular/enzimologia , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/genética , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas de XenopusRESUMO
Ig class-switch recombination (CSR) is directed by the long and repetitive switch regions and requires activation-induced cytidine deaminase (AID). One of the conserved switch-region sequence motifs (AGCT) is a preferred site for AID-mediated DNA-cytosine deamination. By using somatic gene targeting and recombinase-mediated cassette exchange, we established a cell line-based CSR assay that allows manipulation of switch sequences at the endogenous locus. We show that AGCT is only one of a family of four WGCW motifs in the switch region that can facilitate CSR. We go on to show that it is the overlap of AID hotspots at WGCW sites on the top and bottom strands that is critical. This finding leads to a much clearer model for the difference between CSR and somatic hypermutation.
Assuntos
Citidina Desaminase/imunologia , Citidina Desaminase/metabolismo , Switching de Imunoglobulina/fisiologia , Animais , Linfócitos B/enzimologia , Linfócitos B/imunologia , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , DNA/genética , DNA/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Recombinação GenéticaRESUMO
Ig class switch recombination (CSR) and somatic hypermutation require activation-induced cytidine deaminase (AID). The search for AID-interaction factors has been a major research effort in the field, as the mechanism of preferential targeting of AID to Ig loci remains elusive. CTNNBL1 is one of the few identified AID-interacting factors and has been shown to affect AID-mediated mutation and gene conversion in chicken DT40 cells. CTNNBL1 was also implicated in mammalian CSR by the fact that an AID mutant that fails to interact with CTNNBL1 also fails to support CSR in AID-deficient mouse B cells. To directly assess the role of CTNNBL1 in CSR, we disrupted the CTNNBL1 gene on both alleles in mouse CH12F3 cells by gene targeting. We found normal levels of CSR in CTNNBL1-deficient cells, indicating that CTNNBL1 is dispensable for CSR.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Switching de Imunoglobulina/genética , Proteínas Nucleares/fisiologia , Recombinação Genética , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Linhagem Celular Tumoral , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Citidina Desaminase/fisiologia , Marcação de Genes , Imunoglobulina A/genética , Imunoglobulina A/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Recombinação Genética/imunologia , beta CateninaRESUMO
Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1) has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.
Assuntos
Proliferação de Células , DNA Ligases/metabolismo , Deleção de Genes , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , DNA/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/genética , Replicação do DNA , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas de XenopusRESUMO
Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID) that catalyzes numerous DNA cytosine deaminations within switch regions. The resulting uracils are processed by uracil base excision and/or mismatch repair enzymes that ultimately generate switch region DNA double-strand breaks (DSBs). Uracil glycosylase 2 (UNG2) is required for CSR, most likely by removing uracils to generate abasic sites. Although it is presumed that the apurinic/apyrimidinic endonuclease 1 (APE1) generates DNA strand incisions (a prerequisite for CSR) at these abasic sites, a direct test of the requirement for APE1 in CSR has been difficult because of the embryonic lethality of APE1 ablation in mice. Here, we report the successful deletion of the APE1 gene in a mouse B cell line (CH12F3) capable of robust CSR in vitro. In contrast to the general assumption that APE1 is essential for cellular viability, deletion of APE1 in CH12F3 cells has no apparent effect on cell viability or growth. Moreover, CSR in APE1-null CH12F3 cells is drastically reduced, providing direct evidence for an essential role for APE1 in switch region cleavage and CSR. Finally, deletion of AP endonuclease 2 (APE2) has no effect on CSR in either APE1-proficient or -deficient cells.