| Various DNA intermediates generated during the DNA replication and DNA repair processes make the nucleases essential for the maintenance of genome stability. During the past two decades of genome stability research, multiple nucleases have been identified to be required for okazaki fragment maturation, DNA resection and processing of HR intermediates. Despite their importance for the fidelity of DNA reactions, the uncontrolled cleavage of its potential targets such as replication fork, telomere loop and so on, would be detrimental for the completion of chromosome replication and could induce loss of heterozygosity (LOH) and chromosome rearrangement, which is a hallmark of cancer genome. However until now the regulatory mechanism of these nucleases is still largely unknown.Here we identified a novel factor Dsnl as the co-regulator of two different nucleases Rad16-Swi10(XPF-ERCC1in human) and Dna2-Cdc24(Dna2in human) to optimize the DNA replication and repair. Using affinity purification coupled with mass spectrometry analysis, we found that Structure-specific endonucleases Radl6-SwilO, Dna2-Cdc24, Sawl and an unknown protein Spcc1322.02(Dsnl) can form a bigger complex, named "Double Structure-specific Nucleases (DSN) complex". Analysis of complex organization indicates that Dsnl is the mediator between Radl6-Swi10-Sawl and Dna2-Cdc24. Dsn1can interact separately with Rad16-Swi10and Dna2-Cdc24through the middle domain and C terminal domain, while the N terminal domain is required for the cell cycle regulation of its signal in the nucleus. Consistent with their coexist in a bigger complex involved with DNA repair, these factors can be recruited to the double-strand break (DSB) sites in a cooperative manner. The interaction between Rad16-Swi10and Dsnl but not Sawl is required for the cleavage of3’non-homologous tail during the single strand annealing (SSA). In addition, Dsnl is also involved in the removal of covalent Topl-DNA adducts and mating type switch but not nucleotide excision repair (NER). Furthermore our in vitro results show that Dsnl can specifically activate the3’endonuclease activity of Rad16-Swi10.Exol and Rqhl-Dna2-RPA constitute two redundant pathways involved in extensive resection. Dsnl null allel can suppress the resection defect of exol△mutant, which is depends on Rqh1. It indicates that Dsnl inhibit the function of Rqhl-Dna2-RPA pathway in DNA resection. We also found that the inhibitory effect of Dsn1on DNA resection can regulate the choice of donor template during SSA, which may help to maintain the integrity of the genomic fragments between multiple repeats. Consistent with these genetic correlation, Dsnl C terminal fragment purified from E.coli can inhibit the RPA-mediated activation of5’nuclease activity of Dna2-Cdc24complex. Furthermore, we found that the cell cycle regulation of Dsnl through the N terminal domain helps to release the activity of Dna2-Cdc24in S phase. The N terminal truncation or site mutation of Dsnl can cause the abnormal accumulation of Dsnl in S phase, and also enhance cellular sensitivity to DNA damage agents, which can be rescued by the deletion of C terminal domain. In addition, the combination between Dsnl N terminal site mutation with Dna2weak allel, dna2-TAP can cause synthetic lethality. This synthetic lethality can be rescued by the C terminal truncation of Dsnl or pfhl-R20allel. It indicates that the defect induced by misregulation of Dsnl is due to the inhibition of Dna2in DNA repair and replication.In general, our results display that Dsnl can regulate the repair of SSA by activating the3’nuclease activity of Rad16-Swi10and inhibiting the5’nuclease activity of Dna2-Cdc24. We also show that the cell cycle regulation of Dsnl is essential for the optimal function of Dna2-Cdc24and maintenance of genome stability.The research in this paper sheds more light on the novel regulation mechanism of nuc leases, pinpoints the significance of the cell cycle dependent regulation of nucleases, and will promote the investigation about the function of nuclease in maintaining genome stability. |
PDF Full Text Request