Rooney Sean, Alt Frederick W, Sekiguchi JoAnn, Manis John P
Howard Hughes Medical Institute, Children's Hospital, CBR Institute for Biomedical Research, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2471-5. doi: 10.1073/pnas.0409857102. Epub 2005 Feb 7.
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.
B淋巴细胞系细胞中Ig基因的组装涉及两种不同的DNA重排。在早期B细胞发育过程中，种系V、D和J基因片段处的位点特异性双链断裂（DSB）通过非同源末端连接（NHEJ）连接，形成可变区外显子。活化的成熟B细胞可通过类别转换重组（CSR）改变表达的Ig重链恒定区外显子，CSR也涉及DSB中间体。缺乏任何已知的NHEJ因子会严重损害切割后的V、D和J片段的连接。在NHEJ中，由Ku70/80末端结合异二聚体和催化亚基（DNA-PKcs）组成的DNA依赖性蛋白激酶（DNA-PK）激活Artemis，以产生一种在连接前处理DSB的核酸酶。由于DNA-PKcs组分的失活也严重影响CSR，我们测试了DNA-PK是否也通过激活Artemis在CSR中发挥作用。为了避免对V（D）J重组的需求，我们生成了携带预组装的Ig重链和κ轻链“敲入”（HL）等位基因的DNA-PKcs和Artemis缺陷型B细胞。我们发现，Artemis缺陷型HL B细胞经历了强烈的CSR，这表明DNA-PKcs通过一种不依赖Artemis的机制在CSR中发挥作用。为了进一步阐明DNA-PKcs潜在的不依赖Artemis的功能，我们询问在ATM和Artemis双缺陷的小鼠中是否也观察到与DNA-PKcs和相关的共济失调-毛细血管扩张突变（ATM）激酶双缺陷相关的胚胎致死性。我们发现ATM/Artemis双缺陷小鼠是活的，并且以正常的孟德尔数量出生。因此，我们得出结论，DNA-PKcs在CSR和正常发育中具有不依赖Artemis的功能。
