Integration of NHEJ into V(D)J recombination via Ku7080 binding to the RSS heptamer and RAG12

Abstract

The Ku autoantigen (Ku70/80) is a versatile DNA binding protein with prominent DNA-end and sequence-specific binding activities. It participates in the repair of DNA double stranded breaks (DSB) as a component of the nonhomologous end-joining (NHEJ) apparatus, which also includes DNA-PKcs, XRCC4 and DNA ligase N. The Ku autoantigen is also integral to V(D)J recombination, a physiological process whereby the diversity of the immune repertoire is generated by the somatic assembly of the variable (V), diversity (D), or joining (J) gene segments in the immunoglobulin (Ig) or T cell receptor (TCR) variable chain gene in lymphocytes. As a sequence-specific DNA binding protein, the Ku autoantigen is also implicated in transcriptional regulation and control of DNA replication. The overall goal of the present research project was to identify novel specific DNA binding sites for the Ku autoantigen and to link DNA sequence-specific binding by the Ku protein to specific cellular functions. The first principal objective was to directly compare DNA binding activities of Ku70/80 to disparate specific DNA sequences, NRE1 and A3/4, through reciprocal competitive electrophoretic mobility shift assays (EMSAs) and protein-DNA UV-crosslinking studies. The second principal objective was to identify novel classes of Ku70/80-specific DNA binding sites by the systemic evolution of ligands by exponential enrichment (SELEX) with recombinant human Ku70/80, and investigate the functional implications of the interaction of Ku70/80 with the specific DNA binding sites of interest evolved through SELEX. In the present study, the sequence-specific binding activities of the Ku autoantigen to the negative regulatory element 1 (NRE1) in the long terminal repeat (LTR) of the mouse mammary tumor virus (MMTV) and the A3/4 sequence, a sequence homologous to the various mammalian replication origins ( ors), were investigated by reciprocal competitive EMSAs and protein-DNA UV-crosslinking experiments. The results demonstrated that the Ku protein binds to nonspecific DNA ends, NRE1 and A3/4 with apparently distinct affinities. Furthermore, its two subunits were shown to make differential contacts with nonspecific DNA ends, NRE1 and A3/4 in the absence or presence of Mg 2+ and ATP. These findings establish A3/4 as a direct sequence-specific DNA binding site for the Ku autoantigen, which is distinct from either NRE1 or nonspecific DNA ends. (Abstract shortened by UMI.)