Characterization of a novel DNA glycosylase fromS. sahachiroiinvolved in the reduction and repair of azinomycin B induced DNA damage

Abstract

Azinomycin B is a hybrid polyketide/nonribosomal peptide natural product and possesses antitumor activity by interacting covalently with duplex DNA and inducing interstrand crosslinks. In the biosynthetic study of azinomycin B, a gene (orf1) adjacent to the azinomycin B gene cluster was found to be essential for the survival of the producer, Streptomyces sahachiroi ATCC33158. Sequence analyses revealed that Orf1 belongs to the HTH_42 superfamily of conserved bacterial proteins which are widely distributed in pathogenic and antibiotic-producing bacteria with unknown functions. The protein exhibits a protective effect against azinomycin B when heterologously expressed in azinomycin-sensitive strains. EMSA assays showed its sequence nonspecific binding to DNA and structure-specific binding to azinomycin B-adducted sites, and ChIP assays revealed extensive association of Orf1 with chromatin in vivo. Interestingly, Orf1 not only protects target sites by protein–DNA interaction but is also capable of repairing azinomycin B-mediated DNA cross-linking. It possesses the DNA glycosylase-like activity and specifically repairs DNA damage induced by azinomycin B through removal of both adducted nitrogenous bases in the cross-link. This bifunctional protein massively binds to genomic DNA to reduce drug attack risk as a novel DNA binding protein and triggers the base excision repair system as a novel DNA glycosylase.

Structure of a DNA glycosylase that unhooks interstrand cross-links

“…2 B and C). This d7mG activity is modest compared with that of other alkylpurine DNA glycosylases under similar conditions (21,25,26), but demonstrates that an AZB adduct is not necessary for AlkZ activity and corroborates the previous data showing activity toward monoadducts (16). In addition, we found that treatment with NaOH or EndoIV is required to nick the abasic DNA produced by AlkZ, indicating that AlkZ is a monofunctional DNA glycosylase that does not have lyase activity (Fig.…”

“…AlkZ is one of two DNA glycosylases that unhooks an ICL (16,18). The mechanism by which a DNA glycosylase unhooks an ICL is not immediately obvious, because these enzymes typically capture the modified nucleoside inside the active site by extruding it from the DNA helix, and such a base-flipping mechanism would be inhibited by an ICL.…”

“…Previous work has established that AlkZ can unhook an AZB ICL, but whether the enzyme cleaves both sides is less clear (16). Although full ICL excision by a DNA glycosylase would create opposing abasic sites susceptible to a deleterious double-strand break, it also would simplify repair by eliminating the need for NER or TLS/HR pathways to remove the remaining monoadduct.…”

“…In contrast to NER-associated repair of other ICLs, AZB ICLs were recently identified as the target of repair by a DNA glycosylase (16). The azi36 (orf1) gene sits adjacent to the AZB synthesis cluster in S. sahachiroi (17) and provides self-resistance to cells from AZB toxicity.…”

“…The orf1 gene product unhooks AZB ICLs by cleaving the N-glycosidic bond on at least one side of the lesion to produce an abasic site that is recognized by the bacterial AP endonuclease EndoIV (Fig. 1C) (16). Orf1 belongs to the HTH_42 (Pfam 06224) superfamily of uncharacterized winged helix (WH)-containing proteins that exist in various pathogenic and antimicrobial-producing bacteria.…”