: The formation of lymphoid malignancies has been linked to an essential physiological process of lymphocyte development, V(D)J recombination. This recombination proceeds through two steps: a site specific cleavage to generate double stranded DNA breaks, followed by a general repair of these breaks. The latter is carried out by non-homologous end joining (NHEJ) proteins. Mutations in NHEJ genes have been shown to greatly increase genomic instability and oncogenic transformation of lymphocytes. This finding not only indicates the importance of NHEJ proteins in maintaining genome integrity, but also suggests the aberrant nature of an alternative end-joining pathway that may initiate genetic abnormalities. However, little is known about the molecular basis of this aberrant pathway in V(D)J recombination and consequent genetic alterations. Our long term objective is to elucidate the underlying mechanisms of defective V(D)J recombination processes in oncogenic transformation of lymphocytes and to identify potential risk factors that may enhance these processes. In an effort to achieve this goal, we developed recombination-inducible cell lines from one NHEJ-mutant, a severe combined immunodeficient (scid) mouse that bears a mutation in the catalytic unit of the DNA-dependent protein kinase (DNA-PKcs). A large quantity of recombination intermediates can be generated in this scid cell line, and their resolution is conditional, error-prone, and leads to the formation of interlocus recombination products. We propose to extend our work on DNA-PKcs-independent recombination by developing a novel approach to capture any cells that undergo recombination-induced translocation. To further understand the biochemical nature of the aberrant end resolution in scid cells, we will characterize protein structures associated with recombination ends during the course of resolution, and directly test the function of exogenously expressed recombinase on scid coding end resolution. Finally, we will investigate the role of poly(ADP-ribosyl)ation, one of several DNA damage sensing systems, in recognizing, processing and joining recombination ends in these scid cells, as well as in maintaining genome stability.
|Wang, Guannan; Dhar, Kajari; Swanson, Patrick C et al. (2012) Real-time monitoring of RAG-catalyzed DNA cleavage unveils dynamic changes in coding end association with the coding end complex. Nucleic Acids Res 40:6082-96|
|Franco, Daniel; Chang, Yung (2009) Accessibility of chromosomal recombination breaks in nuclei of wild-type and DNA-PKcs-deficient cells. DNA Repair (Amst) 8:813-21|
|Pavlicek, Jeffrey W; Lyubchenko, Yuri L; Chang, Yung (2008) Quantitative analyses of RAG-RSS interactions and conformations revealed by atomic force microscopy. Biochemistry 47:11204-11|
|Zhong, Hanbing; Li, Zhi; Lin, Shuo et al. (2007) Initiation of V(D)J recombination in zebrafish (Danio rerio) ovaries. Mol Immunol 44:1784-92|
|Li, Zhi; Chang, Yung (2007) V(D)J recombination in zebrafish: Normal joining products with accumulation of unresolved coding ends and deleted signal ends. Mol Immunol 44:1793-802|
|Jacobsen, Elizabeth; Beach, Thomas; Shen, Yong et al. (2004) Deficiency of the Mre11 DNA repair complex in Alzheimer's disease brains. Brain Res Mol Brain Res 128:1-7|