Identification of Berenil Target Sites in Plasmid pBR322

Green N,Department Of Biochemistry And Cancer Biology, School Of Medicine, Meharry Medical College, 1005 Db Todd Jr. Bivd, Nashville, Tn, Usa. ,Valenzuela MS

doi:10.19070/2332-2756-170004

Green N, Department Of Biochemistry And Cancer Biology, School Of Medicine, Meharry Medical College, 1005 Db Todd Jr. Bivd, Nashville, Tn, Usa. + Show 1 more

Open Access

https://doi.org/10.19070/2332-2756-170004

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Abstract

Berenil, a minor groove DNA binding molecule, has been extensively used in veterinary medicine. Modeling studies have suggested that berenil binds to A/T rich regions on the DNA and the product of this interaction causes the formation of crosslinks between opposite DNA strands. These crosslinks could potentially inhibit fundamental biological processes including transcription and DNA replication. We had previously used the pBR322 genome as a model system to investigate the role of A/T sequences on berenil activity. We reported that the insertion of poly(dA)poly(dT) sequences into the pBR322 genome causes replication inhibition of the recombinant plasmids when cultures were exposed to berenil. However, we noticed that even in the absence of these sequences the parental plasmid replication was also inhibited, albeit less than the recombinants. This observation led us to the present study were we attempted to identify the location of natural berenil target sites in the pBR322 genome. Through a combination of deletion analysis, recombinant DNA and a replication assay we uncovered a 378 bp DNA fragment that has all the hallmarks of a berenil target site. A recombinant plasmid lacking this region is more refractive to the drug than the parental plasmid, and another variant containing and extra copy of this region increases the susceptibility of the plasmid towards berenil. The 378 bp region is about 60% A/T rich and contains about 21 potential berenil binding sites.

Highlights

Berenil {4-4′-(1-triazene-1,3-dyil)bis(benzamidine)}, an aromatic diamidine [1], displays a trypanocidal, and bactericidal activity, and has been used in veterinary medicine for the treatment of trypanosomiasis [2,3,4]
We observed that even in the absence of the poly(dA)poly(dT) sequence, the parental plasmid itself is sensitive to the drug, natural berenil target sites must be present in the pBR322 genome [17]
This sensitivity was demonstrated by the reduced plasmid yield obtained with both parental and recombinant plasmids, which was shown to be in great in part derived from an inhibition of plasmid DNA replication by the drug [17,18,19]

Summary

Introduction

Berenil {4-4′-(1-triazene-1,3-dyil)bis(benzamidine)}, an aromatic diamidine [1], displays a trypanocidal, and bactericidal activity, and has been used in veterinary medicine for the treatment of trypanosomiasis [2,3,4]. Modeling studies have suggested that berenil binding is through its participation in the formation of hydrogen bond-bridges between opposite A/T nucleotides in the DNA double helix) [9]. These additional hydrogen bonds, could potentially act as inhibitors of processes that fundamentally depend on DNA melting, including transcription and DNA replication. We observed that even in the absence of the poly(dA)poly(dT) sequence, the parental plasmid itself is sensitive to the drug, natural berenil target sites must be present in the pBR322 genome [17]

Methods

Results

Conclusion