Assembly of the nonhomologous end joining machinery.

Abstract

The DNA double strand break (DSB), where the chromosome is physically fragmented, represents a serious threat to the integrity of a genome. Accordingly, two DSB repair mechanisms, homologous recombination (HR) and nonhomologous end joining (NHEJ), are conserved in all kingdoms of life. HR utilizes homologous sequences from sister chromatids to direct DNA synthesis spanning the DSB. In contrast, NHEJ involves direct processing and rejoining of the DSB ends. Because NHEJ plays essential roles in mutagenesis, immunological development and carcinogenesis, a detailed molecular understanding of this process is crucial. This dissertation focuses on determining the forces directing assembly of the NHEJ repair machinery using the robust genetic techniques available in <italic>Saccharomyces cerevisiae.</italic> In this yeast, NHEJ requires the Yku70-Yku80 (Ku), Mre11-Rad50-Xrs2 (MRX) and the Dnl4-Lif1-Nej1 (DNA ligase IV) protein complexes. We hypothesized that NHEJ requires contacts between these complexes to coordinate repair. A yeast two-hybrid screen of NHEJ proteins identified interactions between Yku80-Dn14, Xrs2-Lif1 and Mre11-Yku80. The Yku80-Dn14 interaction was refined to the Yku80 C-terminus and mutational analysis identified three conserved leucines, part of a putative amphipathic alpha helix, which determined interaction with Dn14. Mutation of the Yku80 C-terminus resulted in moderate NHEJ defects. The Xrs2-Lif1 interaction was refined to the Xrs2 fork-head associated (FHA) domain and the Lif1 C-terminus. Mutation of Xrs2 identified four residues, expected to contact phosphothreonines, which mediate interaction with Lif1. Correspondingly, two Lif1 threonines, T387 and T417, mediated interaction with Xrs2. Mutation of the Xrs2 FHA domain caused slight defects in NHEJ, but specifically and severely blocked NHEJ and recruitment of Dnl4 to a DSB when combined with the Yku80 C-terminal mutations. Overall, this work describes redundant interactions of DNA ligase IV with Ku and MRX that provide a framework for understanding assembly of the NHEJ machinery at a DSB. Finally, we demonstrated that the Mt-Ku and Mt-Lig proteins from the bacteria, <italic>Mycobacterium tuberculosis,</italic> are sufficient to reconstitute NHEJ in yeast, indicating that they represent a <italic>bona fide</italic> NHEJ system. Characterization of this simple system consisting of only two polypeptides provides a further model for exploration of the rules governing assembly of the NHEJ machinery.