Characterization Of A Novel Dna Glycosylase Involved In The Repair Of Azinomycin B Induced Dna Damage

Azinomycin B is a hybrid polyketide/nonribosomal peptide natural product and possesses antitumor activity by interacting covalently with duplex DNA in the major groove and inducing interstrand crosslinks.Initial cytotoxicity studies and early phase clinical investigation showed that azinomycin B is highly active at the nanomolar level.Therefore,the intensive investigation of azinomycin B biosynthesis is of great significance in the development of chemotherapeutic agents.The azinomycin B biosynthetic gene cluster in S.sahachiroi was reported in 2008.Its biosynthetic pathway,however,is still not completely understood.To date,only the enzymatic cascade leading to the formation of the naphthoate moiety has been unequivocally deciphered.Owing to existence of many novel genes and lack of candidates for self-resistance and regulation within the published gene cluster,analysis and characterization of correlative genes will contribute to the elucidation of molecular mechanisms of its biosynthesis,self-resistance and regulation.In this study,we focused on genes of unknown function within or close to the putative azinomycin B gene cluster,and successfully constructed in frame deletion mutant strains of 17 genes.The analyses of their fermentation products indicated that deletion of 7 of them greatly affected the production of azinomycin B.The corresponding complementation strains restored the ability to produce azinomycin B at a certain level,which confirmed the involvement of these genes in the azinomycin B biosynthesis.Moreover,production of azinomycin B as well as naphthoate intermediate decreased dramatically in the gene deletion mutant of aziG encoding a thioesterase.This result implied that AziG might be responsible for the release of naphthoate.The mutant △aziD1 did not produce azinomycin B but yielded trace amount of active compounds at the same retention time during HPLC analyses,which suggested a possible involvement of AziD1 in the post-NRPS modification of pre-azinomycin B.In addition azinomycin B biosynthesis was totally abolished when aziU1,aziU2,aziU3,aziD2 or aziD3 was absentIn the gene deletion experiments,a gene（orf1）adjacent to the right boundary of the gene cluster,could not be deleted from its natural chromosomal context until a second copy of the gene had been introduced into the chromosome at a different location.The result indicated that orf1 is essential for the wild type strain.However,it became easy to eliminate orf1 in the non-azinomycin B-producing mutant △aziU3,implying that the essential nature of orf1 is closely bound to production of azinomycin B.The Orf1 protein exhibits a protective effect against azinomycin B when heterologously expressed in azinomycin-sensitive strains.Moreover,compared to the original strain △aziU3,the orf1 deletion mutant strain △aziU3Aorf1 exhibited increased sensitivity to azinomycin B,which demonstrated the resistance function of Orf1 in the native host.Gel mobility shift assays（EMSA）showed that Orf1 binds sequence nonspecifically to native DNA and structure-specifically to azinomycin B-modified sites,and ChIP assays revealed extensive association of Orf1 with chromatin in vivo.Interestingly,in vitro DNA protection assays revealed Orf1 not only protects target sites by protein-DNA interaction but is also capable of repairing azinomycin B-mediated DNA cross-linking.A series of DNA repair assays in vitro undoubtedly demonstrated that the cross-linking of azinomycin-modified DNA was reversed by Orf1.In view of its cleavage effect on the crosslinked DNA,Orf1 was supposed to repair azinomycin B-adducted DNA by excision of damaged bases.Therefore,we used Endo Ⅳ,an AP endonuclease which specifically recognizes and cleaves abasic DNA,to confirm the glycosylase activity of Orf1,and found that numerous AP sites were produced by Orf1 in DNA strands of the azinomycin-DNA adducts.Further intensive DNA cleavage assays were conducted by using a 21-mer DNA duplex containg one crosslinking site as the substrate,the result of which proved that Orfl hydrolyzes the glycosidic bonds of both purines in the crosslink without bias.It is a peculiar DNA glycosylase that specifically recognizes interstrand azinomycin B-DNA crosslinks and removes modified bases located on the two DNA strands,forming apurinic（AP）sites and triggering the base excision repair system.The novel DNA glycosylase,Orf1,confers resistance in azinomycin B biosynthesis via distinct DNA binding ability and DNA repair capability.Sequence analyses revealed that Orf1 belongs to the HTH₄2 superfamily of conserved bacterial proteins which are widely distributed in pathogenic and antibiotic-producing bacteria with unknown functions.Orfl is the first member of this superfamily to have a defined biological function in bacterial self-resistance identified.Moreover,this is the first time that a DNA glycosylase is found to be employed in the self-defence to a dual alkylating agent.The results of this study enlarge the scope of the base-excision repair pathway in microbial secondary metabolism and provide new insight into the biological study of the newly found HTH₄2 superfamily proteins.