Improperly repaired DNA double-strand breaks (DSBs) can lead to cell death or chromosomal rearrangements that contribute to oncogenic transformation. The two major mammalian DSB repair pathways are homologous recombination (HR), which is active in post-replicative (S/G2) cells, and classical non-homologous end joining (C-NHEJ), which predominates in pre-replicative (G1) cells. For C-NHEJ, the Ku70/Ku80 heterodimer (""""""""Ku"""""""") recognizes DSBs and the XRCC4/Ligase4 (Lig4) complex joins them. During V(D)J recombination in developing B lymphocytes, RAG endonuclease generated DSBs at V, D, and J gene segments are joined exclusively by C-NHEJ to assemble exons encoding antigen receptor/antibody variable regions. During immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) in activated mature B lymphocytes, activation-induced cytidine deaminase (AID)-initiated DSBs in IgH switch (S) regions are fused, predominantly by C-NHEJ, to exchange expressed IgH constant region exons. Aberrant joining of V(D)J- or CSR-associated DSBs can lead to chromosomal translocations that fuse antigen receptor loci to oncogenes, and, thereby, contribute to lymphomagenesis. In the absence of C-NHEJ, RAG- or AID-initiated IgH DSBs can be joined to form such oncogenic chromosomal translocations by an alternative end-joining (A-EJ) pathway (or pathways). In addition, CSR is carried out relatively robustly by A-EJ in the absence of Lig4, Ku, or both. Based on substantial preliminary data, we propose the A-EJ in the absence of Lig4 is distinct from that which occurs in the absence of Ku (or Ku plus Lig4). We refer to these two A-EJ pathways as """"""""Lig4-independent"""""""" and """"""""Ku- independent"""""""" A-EJ, respectively. We propose to elucidate DSB recognition and joining components of the two A-EJ pathways, their relative activity in the G1 cell cycle, and their relative contributions to normal versus aberrant end-joining that promotes chromosomal translocations. Specifically, we propose to 1) Identify factors that mediate Lig4- and/or Ku-independent A-EJ during CSR and 2) Assess potential roles of Lig4- and/or Ku- independent A-EJ in G1-arrested pro-B lines.
Our genome is constantly threatened by DNA damage, the most severe of which is a DNA Double Strand Break (DSB). Our lab recently identified an ill-defined Alternative End-Joining pathway to repair DSBs, which has been thought to catalyze potentially dangerous genomic rearrangements, such as translocations, which may lead to, for example, cancer. To gain fundamental insight into how we maintain genomic stability, this proposal will thoroughly characterize Alternative End-Joining in DNA break repair and translocation formation.
|Kumar, Vipul; Alt, Frederick W; Frock, Richard L (2016) PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line. Proc Natl Acad Sci U S A 113:10619-24|