Here, we report the first systematic dissection of how ICL repair might operate in the Trypanosoma brucei, the causative agent of African trypanosomiasis, and demonstrated that this diverged eukaryote expresses systems that show some intriguing differences to those mechanisms present in other organisms. To combat their deleterious effects all eukaryotes have developed cell cycle-dependent repair strategies that co-opt various factors from вЂclassicalвЂ™ DNA repair pathways to resolve such lesions. Interstrand crosslinks (ICLs) represent a highly toxic form of DNA damage that can block essential biological processes including DNA replication and transcription. Taken together, these findings suggest that SUMOylation of Lif1 represents a new regulatory mechanism that downregulates NHEJ in a cell cycle phase-independent manner. This is likely due to the inhibitory effect of Lif1 SUMOylation on both its self-association and newly observed single-strand DNA binding activity. The lif1-K301R mutant cells exhibit increased levels of NHEJ repair compared with wild-type cells throughout the cell cycle. We identify lysine 301 as the major conjugation site and demonstrate that its replacement with arginine completely abolishes Lif1 SUMOylation in vivo and in vitro. Single-strand DNA, but not double-strand DNA or the Lif1 binding partner Nej1, is in-hibitory to Lif1 SUMOylation. We show that Lif1 SUMOylation occurs throughout the cell cycle and requires the Siz SUMO ligases.

Here, we identify and characterize the SUMOylation of budding yeast Lif1 protein, which is required for the ligation step in NHEJ. The mechanisms regulating NHEJ efficiency and interplay between its components are not fully understood. Non-homologous end-joining (NHEJ) repairs DNA double-strand breaks by tethering and ligating the two DNA ends. Instead, its function in this process seems to be rather individual. Here we show that Pso2 associates with none of the above DSB repair proteins, suggesting that this protein very likely does not act in the repair of ICL-associated DSB via crosstalk with DSB repair machinery. In order to address the question whether function of Pso2 in the repair of ICL-associated DSB could be mediated through protein-protein interactions, we have conducted a comprehensive two-hybrid screen examining a possibility of interaction of Pso2 with Yku70, Yku80, Nej1, Lif1, Dnl4, Rad50, Mre11, Xrs2, Rad51, Rad52, Rad54, Rad55, Rad57, Rad59 and Rdh54. Phenotype of the pso2 mutant suggests that Pso2 is not involved in incision step of ICL repair, but it rather functions in some downstream event such as processing of DNA ends created during generation of ICL-associated double-strand breaks (DSB). These have been designated PSO1-10 based on the psoralen sensitive phenotype of the corresponding mutants. Moreover, there are additional factors significantly influencing the repair of ICL in this organism. In Saccharomyces cerevisiae, ICL repair utilizes factors from all of the three major repair groups: nucleotide excision repair (RAD3 epistasis group), post-replication repair (RAD6 epistasis group) and recombinational repair (RAD52 epistasis group). Therefore, they present a colossal challenge for the DNA damage response and repair pathways. DNA interstrand cross-links (ICL) are thought to be one of the most lethal forms of DNA damage.

Pso2 gamma control