Invesgating The Biological Function Of Three Genes Located Downstream To The Distamycin Biosynthetic Gene Cluster: Dst27,dst28 And Dst29

Distamycin is a polyamide antibiotic and is produced by Streptomyces netropsis.It has good anti-tumor potency.The structure of distamycin features an oligopeptidic pyrrolecarbamoyl unit enabling it to bind reversibly to the AT rich region of B DNA minor groove through non-covalent bond and thus block the DNA template activity.Based on this,DST has been developed as sequence specific drug to treat cancer.Our research focus on 3 overlooked immediate down stream genes of distamycin biosynthesis gene cluster,namely dst27,dst28,dst29.By using bioinformatic tools,molecular research methods comprehensively,we found that dst27 is a P4 family primase and dst28 is a replication initiation protein with a wingled helix DNA binding domain while dst29 is a helicase which belongs to the Uvr D family.Knoncking out dst27,dst28,dst29 decrease the yeild of distamycin while over-expressing these genes in combination or separately brings silimar influence to the yield of distamycin both in vivo and in vitro.Considering that distamycin is a typical DNA binding antibiotic and the self-resistance mechanism of the DNA binding antibiotic family usually contains a set of DNA repair/replication related genes,we think that dst27,dst28,dst29 may invoveld in the self-resitsance mechanism of S.netropsis.To validate our hypothesis,we conduct the heterologous expression of dst27,dst28,dst29 in Streptomyces lividans TK24 and performed the disk diffusion assay to detect the change of distamycin resistance level.We found out that heterologous express these 3 genes in S.lividans,no matter in combination or separately will bring a significant change in the distamycin resistance level.Resistance level raise significantly in dst28::dst29heterologous expression strain.This confirm our hypothesis that dst27,dst28,dst29 indeed take part in the self-resistance of S.netropsis.We find out that the homologs of dst27,dst28,dst29 exist widely in other DNA binding agents producers.For instance the mitomycin producer Cross resistance assay of mitomycin show that dst27,dst28,dst29 could also react to mitomycin.Therefore these 3 genes may represent a common self-resistance mechanism in DNA binding antibiotic producers.In conclusion,we found that dst27,dst28,dst29 are involved in the self-resistance mechanism of S.netropsis.These genes may also represent a common and novel selfresistance mechanism existing in many DNA damage/binding agent producers.