A Functional And Mechanistic Study For Slx4-Rtt107 Complex In DNA Repair By Non-Homologous End Joining

DNA double-strand breaks(DSBs)are repaired by two main pathways,homologous recombination(HR)and non-homologous end joining(NHEJ).Although the mechanism of homologous recombination is well-characterized,the molecular details of non-homologous end joining are not well understood.NHEJ includes two subpathways,i.e.,precise and imprecise end-joining.Here,We investigated the roles of scaffold proteins Slx4 and Rtt107 in NHEJ repair,and obtained the following results.1)We show that the Slx4 and Rtt107 scaffolds form a complex that participates in the imprecise NHEJ pathway.Using a chromosome-based DSB induction system,we found that deletion of SLX4 or RTT107 significantly reduced the efficiency of imprecise but not precise end-joining,although these mutants displayed only a minor effect on a plasmid-based repair assay.The slx4 mutant that fails to interact with Rtt107,but retains other known functions,is defective in imprecise end-joining on chromatin context,indicating that Slx4 interaction with Rtt107 is important for an efficient NHEJ.2)By using chromatin immunoprecipitation(ChIP)assay,we observed robust Slx4 and Rtt107 recruitment to DSBs,suggesting that Slx4-Rtt107 may promote NHEJ directly.Next,we found that deletion of SLX4 or RTT107 does not affect the recruitment of core NHEJ proteins,such as,Mre11 Yku70,Dnl4 and Pol4 to DSBs.This suggests that the reduced NHEJ efficiency in slx4? or rtt107? mutant was not due to the defective assembly of core NHEJ proteins at DSBs.3)Further,we observed that the sequences for the products repaired by imprecise NHEJ are altered in slx4? or rtt107? mutant when compared to that in WT cells.In wild-type cells,15%of the products repaired by imprecise NHEJ carries insertions and 85%of products carries deletions at junction regions.In contrast,in slx4? or rtt107?mutant,up to over 80%of repair products contain insertions while only approximately 15%of products contain deletions events.These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that the outcomes are affected by Slx4 and Rtt107.4)Further analysis of the repaired products suggested a potential functional relationship between Slx4-Rtt107 and end-processing factors.Radl,which forms a heterodimeric structure-specific endonuclease complex with Radl0,acts to remove 3'flap during SSA and MMEJ.In cells lacking Rad1,we observed a sharp increase for the repair products carrying insertions and consequently a decrease for the products carrying deletions,which likely result from aberrant removal of 3'-flap.Importantly,the double mutants radl?slx4? or radl?rtt107? are as defective as radl?,slx4?,or rtt107? single mutant in 3'-flap removal,suggesting that these genes act in the same pathway for flap removal.5)As seen in the SSA model,we found that the scaffold protein Sawl is also involved in the Radl-dependent 3'-flap removal during NHEJ and SAW1 acts in the same pathway with SLX4,RTT107 and RADl.6)By monitoring the recruitment of Rad1,we found that deletion of SLX4,RTT107 or SAW1 significantly reduced the recruitment of Rad1.The results strongly suggest that Slx4-Rtt107 and Sawl play key roles in targeting Rad1 to NHEJ intermediates with protruding 3'-flap.7)Moreover,deletion of SLX4 or RTT 107 significantly reduced the recruitment of Sawl to DSBs.Thus,it appears that Slx4-Rtt107 targets Sawl to DSBs,and Sawl facilitates the targeting of Radl at 3' tailed substrates to process 3'-flaps.Consistently,the sawl mutant deficient in Sawl-Rad1 interaction,or the slx4 mutant that fails to interact with Rtt107 both displayed reduced Rad1 recruitment.The results suggest that the physical interaction between Sawl and Rad1,Slx4 and Rtt107 is important in tethering Rad1 to 3,tails.Together,we propose that the ordered assembly of the Slx4-Rttl 07,Sawl and Rad1-Rad10 cleavage complex is critically important to remove the 3'-nonhomologous tails formed during imprecise NHEJ.Loss of any single factor among the complex can result in decreased binding of Rad1 to a DSBs and deficient 3'-nonhomologous tail removal.This regulation allows Rad1 to be tightly regulated such that the processing of 3'-flap can be fine-tuned to match the extent and type of end-processing.Our findings reveal that these end-processing factors play important roles in controlling the flexibility and repair outcomes of NHEJ.