DrrC protein of Streptomyces peucetius removes daunorubicin from intercalated dnrI promoter

Abstract

DrrC is a DNA-binding protein of Streptomyces peucetius that provides self-resistance against daunorubicin, the antibiotic produced by the organism. DrrC was expressed in E.coli and purified by using N-terminal MBP-tag which retained DNA-binding property in spite of the tag. Mobility shift assay confirmed the interaction of 313bp DNA that has the dnrI promoter, daunorubicin and MBP-DrrC in the presence of ATP. Biotinylated and immobilized 313bp DNA was intercalated with daunorubicin to observe the release of the drug when MBP-DrrC is allowed to act on the DNA. The release of daunorubicin was recorded by absorption and fluorescence spectroscopy. The experiments proved that daunorubicin was released from DNA in the presence of MBP-DrrC. Fluorescence emission of daunorubicin had a maximum peak at 591nm. However, emission spectrum of released daunorubicin showed hypochromism with a maximum peak at 584nm that is possibly because it is in complex with MBP-DrrC. We propose that DrrC naturally binds at intercalated sites to eject daunorubicin; in the process both drug and protein are dislodged from DNA. Like UvrA, DrrC possibly scans the DNA for intercalated daunorubicin. When it encounters daunorubicin, DrrC dislodges it, thereby allowing DNA replication and transcription to go on unhindered. Thus a novel self resistance mechanism by DNA repair is mediated by DrrC.

p-Aminophenylalanine Involved in the Biosynthesis of Antitumor Dnacin B1 for Quinone Moiety Formation

“…The dnacin B1 BGC contained three regulator genes, separately belonging to the TetR family ( dinR2 ), SARP family ( dinR3 ) and LysR family ( dinR9 ). DinR8, annotated as a DNA excision repair enzyme UvrA, showed 84% similarity with NapR1, and was deduced as the resistance gene in dnacin B1 biosynthesis [ 34 ]. In addition, five ABC transporter genes, dinR1 and dinR4-7 were also found in dnacin B1 BGC ( Table 1 ).…”

Antibiotic Resistance Mechanisms in Bacteria: Relationships Between Resistance Determinants of Antibiotic Producers, Environmental Bacteria, and Clinical Pathogens

“…Antibiotic removal from the target site provides another protective resistance mechanism. In S. peucetius , DrrC removes intercalated daunorubicin/doxorubicin from DNA resulting in normal transcription and replication ( Prija and Prasad, 2017 ). In S. rimosus , the antibiotic oxytetracycline is removed by OtrA from the ribosome ( Doyle et al, 1991 ; Mak et al, 2014 ).…”

“…Most producer organisms contain several mechanisms for self-resistance. For example, S. peucetius relies on DrrAB to efflux doxorubicin ( Li et al, 2014 ; Brown et al, 2017 ), DrrC to remove the antibiotic from its target DNA ( Prija and Prasad, 2017 ), and DrrD is possibly used to modify the antibiotic to an inactive form ( Karuppasamy et al, 2015 ). In addition, there is also a serine protease capable of sequestering daunorubicin to prevent its re-entry into the cell following efflux ( Dubey et al, 2014 ).…”

Antibiotic export: transporters involved in the final step of natural product production

“…After making a double drrAB mutant, which proved more sensitive to exogenous dauno-/ doxorubicin and also produced less antibiotic, Srinivan and colleagues [53] connected the observed phenotype to a feedback transcriptional regulatory mechanism, where deficient export has a part in downregulating some of the biosynthetic genes (we will see more examples of regulatory roles below). The authors reported they could not produce a triple deletion mutant lacking drrAB and drrC [53], where DrrC is UvrA-like additional resistance factor [54]. Since individual drrAB and drrC deletions could be made in S. peucetius, the authors argued for a cooperative role of DrrAB and DrrC in self-immunity [53].…”