B-Z junctions in supercoiled pRW751 DNA contain unpaired bases or non-Watson-Crick base pairs

Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA, but only the latter requires non-paired bases for the reaction. Nuclease S1 and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the "outer" boundaries between the (dC-dG)n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751. As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease S1 sensitive sites. The results suggest that the "outer" B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson-Crick base pairs.     

Theoretical Study of Ethidium Intercalation in Triple-Stranded DNA and at Triplex-Duplex Junctions

Abstract

The contribution of different factors in the interaction of ethidium intercalated into various sequences of a triple helix, or in the region of the junction between the double- and triple-stranded DNA has been studied by energy minimization. It is found that in the total energy of the ethidium - triple helix complexes, a particular electrostatic contribution emerges due to the presence of protonated cytosines in the triple helix. This parameter is determinant in the sequence-specificity of ethidium binding to the triple helix. The preferred intercalation sites of ethidium in the triple helix are proposed. The interaction of ethidium at the triplex-duplex junction, and its effects are also discussed. This study is aimed at searching for new drugs specific for the triple helix, or for the triplex-duplex junctions.     

Screening of promising molecules against MurG as drug target in multi-drug-resistant-Acinetobacter baumannii - insights from comparative protein modeling,molecular docking and molecular dynamics simulation

Abstract

The UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (MurG) is located in plasma membrane which plays a crucial role for peptidoglycan biosynthesis in Gram-negative bacteria. Recently, this protein is considered as an important and unique drug target in Acinetobacter baumannii since it plays a key role during the synthesis of peptidoglycan as well as which is not found in Homo sapiens. In this study, initially we performed comparative protein modeling approach to predict the three-dimensional model of MurG based on crystal structure of UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (PDB ID: 1F0K) from E.coli K12. MurG model has two important functional domains located in N and C- terminus which are separated by a deep cleft. Active site residues are located between two domains and they are Gly20, Arg170, Gly200, Ser201, Gln227, Phe254, Leu275, Thr276, and Glu279 which play essential role for the function of MurG. In order to inhibit the function of MurG, we employed the High Throughput Virtual Screening (HTVS) and docking techniques to identify the promising molecules which will further subjected into screening for computing their drug like and pharmacokinetic properties. From the HTVS, we identified 5279 molecules, among these, 12 were passed the drug-like and pharmacokinetic screening analysis. Based on the interaction analysis in terms of binding affinity, inhibition constant and intermolecular interactions, we selected four molecules for further MD simulation to understand the structural stability of protein-ligand complexes. All the analysis of MD simulation suggested that ZINC09186673 and ZINC09956120 are identified as most promising putative inhibitors for MurG protein in A. baumannii.

Communicated by Ramaswamy H. Sarma