Artemis-independent functions of DNA-dependent protein kinase in Ig heavy chain class switch recombination and development.
Assembly of Ig genes in B lineage cells involves two distinct DNA rearrangements. In early B cell development, site-specific double strand breaks (DSBs) at germ-line V, D, and J gene segments are joined via nonhomologous end-joining (NHEJ) to form variable region exons. Activated mature B cells can change expressed Ig heavy chain constant region exons by class switch recombination (CSR), which also involves DSB intermediates. Absence of any known NHEJ factor severely impairs joining of cleaved V, D, and J segments. In NHEJ, DNA-dependent protein kinase (DNA-PK), which is comprised of the Ku70/80 end-binding heterodimer and the catalytic subunit (DNA-PKcs), activates Artemis to generate a nuclease that processes DSBs before ligation. Because inactivation of DNA-PKcs components also severely affects CSR, we tested whether DNA-PK also functions in CSR via activation of Artemis. To obviate the requirement for V(D)J recombination, we generated DNA-PKcs- and Artemis-deficient B cells that harbored preassembled Ig heavy chain and kappa-light chain "knock-in" (HL) alleles. We found that Artemis-deficient HL B cells undergo robust CSR, indicating that DNA-PKcs functions in CSR via an Artemis-independent mechanism. To further elucidate potential Artemis-independent functions of DNA-PKcs, we asked whether the embryonic lethality associated with double-deficiency for DNA-PKcs and the related ataxia-telangiectasia-mutated (ATM) kinase was also observed in mice doubly deficient for ATM and Artemis. We found that ATM/Artemis double-deficient mice were viable and born in normal Mendelian numbers. Therefore, we conclude that DNA-PKcs has Artemis-independent functions in CSR and normal development.
Rooney
Sean
S
Howard Hughes Medical Institute, Children's Hospital, CBR Institute for Biomedical Research, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Alt
Frederick W
FW
Sekiguchi
JoAnn
J
Manis
John P
JP
D000483:Alleles; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases; D017578:Immunoglobulin Class Switching; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007134:Immunoglobulin Switch Region; D051379:Mice; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D025521:Tumor Suppressor Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004720:Endonucleases; C473337:Dclre1c protein, mouse