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Frederick W. Alt
Children’s Hospital and the Center for Blood Research
Harvard Medical School
Boston, MA
Generally Expressed Gene Products Involved in V(D)J Recombination and DNA Repair
V(D)J recombination is initiated by the lymphoid-specific RAG-1 and RAG-2 proteins that recognize recombination signal sequences (RS) that flank each V, D, and J segment and then introduce double strand breaks (DSBs) between the gene segments and flanking RS, resulting in blunt 5’-phosphorylated recombination signal (RS) ends and covalently-sealed hairpin coding ends. Joining of RS and coding ends relies on generally-expressed cellular factors involved in DNA double strand break (DSB) repair (DSBR).
Ku is a heterodimer of 70kDa (Ku70) and 80kDa (Ku80) subunits which binds to DSBs. DNA end-bound Ku can associate with the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and, thereby, activate its kinase activity. The Ku80 and DNA-PKcs gene products were found to be involved in VDJ recombination/DSBR reaction based on their mutation in ionizing-radiation sensitive mutant cell lines and/or Scid mice. We showed that Ku70 is similarly involved by generating and analyzing Ku70-/-ES cells and mice. Together, our findings support the notion that Ku may serve as a sensor of DSBs in the context of DSBR mechanisms that employ DNA-PK, including that incorporated into V(D)J recombination.
However, the precise role of Ku and DNA-PK in the V(D)J and DSBR reactions remains to be elucidated. Through its intrinsic activities, Ku may function in DNA-end processing (e.g. end-production or un-winding); on the other hand, Ku may also function by recruiting DNA-PKcs, which then may recruit or disassemble other factors involved in the reaction. Notably, Ku70-deficiency, Ku 80 deficiency and the homozygous scid mutation (which alters DNA-PKcs) have overlapping but unique phenotypes in mice, suggesting the possibility of some distinct functions for the three DNA-PK components.
The notably less-severe impact of the scid (DNA-PKcs) mutation suggests independent (from putative role via DNA-PK) functions for Ku proteins in the reaction and/or incomplete DNA/PKcs inactivation by the scid mutation. We have completely inactivated the DNA-PKcs gene in ES cells to distinguish these possibilities. We also identified a ubiquitously expressed gene product we termed XRCC4 that is involved in the DSBR/VDJ reaction. The XRCC4 gene encodes a 334aa nuclear protein with no overall homology to any known protein. Others recently showed that XRCC4 strongly associates with DNA ligase IV suggesting that XRCC4 may function to recruit DNA ligase IV to complete the end-joining reaction. Recently, we have generated XRCC4 KO mice and showed that they have unique phenotype as compared to Ku70/80 KO or Scid mice, indicating that XRCC4 has additional functions beyond those carried out with Ku/DNA-PK.
We will discuss recent efforts to generate and characterize double knock-out (DKO) cells and mice for XRCC4, DNA-PKcs, and ligase IV with respect to potential functions of these components.
References:
1. Li, Z., Otevrel, T., Gao, Y., Cheng, H.-L., Seed, B., Stamato, T.D., Taccioli, G.E., and Alt, F.W. (1995) The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell 83, 1079-1089.
2. Gu, Y., Jin, S., Gao, Y., Weaver, D.T., and Alt, F.W. (1997) Ku70-deficient ES cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination. Proc. Natl. Acad. Sci. USA 94, 8076-8081.
3. Gu, Y., Seidl, K.J., Rathbun, G.R., Zhu, C., Manis, J.P., van der Stoep, N., Davidson, L., Cheng, H.-L., Sekiguchi, J.M., Frank, K., Stanhope-Baker, P., Schlissel, M.S., Roth, D.B., and Alt, F.W. Growth retardation and leaky SCID phenotype of Ku70 deficient mice. (1997) Immunity 7, 653-665.
4. Mizuta, R., Cheng, H.-L., Gao, Y., and Alt, F.W. (1997) Molecular genetic characterization of XRCC4 function. Int. Immunol. 9, 1607-1613.