Karen Frank, M.D., Ph.D.

Molecular Basis of Antigen Receptor Formation in Lymphocytes and Mechanism of DNA Double Strand Break Repair

Research Summary

V(D)J Recombination and DNA Repair
V(D)J recombination is a tightly regulated DNA rearrangement process required to generate a specific immune response through the creation of diverse antigen receptors. The antigen-specific region of the receptor is generated by the assembly of composite gene segments using a site-directed, non-homologous recombination reaction. V(D)J recombination is initiated by the lymphoid-specific RAG endonuclease by the generation of specific double-strand breaks. Next, the nonhomologous DNA end joining (NHEJ) pathway is utilized to complete the V(D)J recombination process.

Nonhomologous End Joining
The NHEJ pathway is not specific to the V(D)J recombination pathway, but is one of the major pathways of DNA repair. DNA in any cell, not just blood cells, can be damaged from exposure to radiation or environmental chemicals, or from the by-products of metabolism. These DNA breaks are potentially harmful to the cell and must be repaired for the cell to survive. Five proteins in this pathway have been identified, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the Ku70/80 heterodimer (Ku), and the XRCC4 and DNA ligase IV proteins. XRCC4 and DNA ligase IV form a complex to complete the final ligation step of the repair process.

Deficiencies of Proteins in the NHEJ Pathway
Deficiencies of Ku 70, Ku 80, XRCC4, or DNA ligase IV lead to a similar phenotype, including growth defects, premature senescence, sensitivity to ionizing radiation, and defective V(D)J recombination. Deficiency of either XRCC4 or DNA ligase IV in mice also causes embryonic lethality. The mutant embryos exhibit defective neurogenesis manifested by extensive apoptosis of newly generated postmitotic neurons. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins, and point to the general stage of neuronal development in which these proteins are necessary. Studies involving the cross of DNA ligase IV-deficient mice with p53-deficient mice (a cancer prone mouse strain), demonstrated that the ligase IV deficiency shortened the latency of tumor development and changed the type of lymphoma that the mice develop.

Association of V(D)J Recombination and NHEJ with Human Disease
Genetic defects in factors involved in this pathway can lead to severe immunodeficiency, resulting in an inability to fight infections. Errors in the V(D)J recombination mechanism can lead to chromosomal rearrangements that lead to cancer. Also, defects in NHEJ are associated with genetic instability which has been associated with cancer development.

Research Questions Related to V(D)J Recombination and Nonhomologous End Joining
Are there additional proteins involved in the NHEJ complex? How do the known proteins interact with one another? What is the role of the BRCT (BRCA1 C-terminal homology) domains of DNA ligase IV in these interactions? How are the XRCC4 and DNA ligase IV proteins regulated during this process?

Selected Papers

*Frank KM, *Sekiguchi JM, Seidl KJ, Swat W, Rathbun GA, Cheng H-L, Davidson L, Kangaloo L, and Alt FW. (1998). Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature,396:173-177. *These authors contributed equally.

Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJ, Sekiguchi JM, Rathbun GA, Swa W, Wang J, Bronson RT, Malynn BA, Bryans M, Zhu C, Chaudhuri J, Davidson L, Ferrini R, Stamato T, Orkin SH, Greenburg ME, and Alt FW. (1998). A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis. Cell, 95:891-902.

Gu Y, Sekiguchi J, Gao Y, Dikkes P, Frank K, Ferguson D, Hasty P, Chun J, and Alt FW. (2000). Defective embryonic neurogenesis in Ku-deficient but not DNA-dependent protein kinase catalytic subunit-deficient mice. Proc. Natl. Acad. Sci USA, 97:2668-2673.

Ferguson DO, Sekiguchi JM, Chang S, Frank KM, Gao Y, DePinho RA, and Alt FW. (2000). The non-homologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations. Proc. Natl. Acad. Sci. USA, 97:6630-6633.

*Frank KM, *Sharpless NE, Gao Y, Sekiguchi JM, Ferguson DO, Zhu C, Manis JP, Horner J, DePinho RA, and Alt FW. (2000). DNA ligase IV-deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway. Molec. Cell, 5:993-1002. *These authors contributed equally.

Wang YG, Nnakwe C, Lane WS, Modesti M, Frank KM. (2004). Phosphorylation and regulation of DNA ligase IV stability by DNA-dependent protein kinase. J Biol Chem. 2004 Sep 3;279(36):37282-90. Epub 2004 Jun 11.

Foster, R.E, Nnakwe, C, Woo, L, and Frank, K.M., Monoubiquitination of the nonhomologous end joining protein XRCC4. Biochem Biophys Res Commun. 341:175-183, 2006.

Woo, L.L., Futami, K., Simamoto, A., Furuichi, Y., and Frank, K.M., The Rothmund-Thomson Gene Product, RECQL4, Localizes to the Nucleolus in Response to Oxidative Stress. Exp. Cell Res. 312:3443-3457, 2006.