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J. Bacteriol. doi:10.1128/JB.01155-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

DNA bending in the mycobacterial plasmid pAL5000 origin-RepB complex

Bose Institute, Dept. Of Microbiology, P1/12 C.I.T. Scheme VIIM, Kolkata 700054, India

^* To whom correspondence should be addressed. Email: sujoy{at}boseinst.ernet.in.

	Abstract

Plasmid pAL5000 represents a family of relatively newly discovered cryptic plasmids in Gram-positive Actinomycetes bacteria. Replication regions of these plasmids comprise of a bi-cistronic operon repA-repB, encoding two replication proteins. Located upstream is a cis acting element that functions as the origin of replication. It comprises of a 200 bp segment spanning two binding sites for the replication protein RepB - a Low affinity, L and a high affinity H separated by a 40 bp spacer sequence. The trajectory of the DNA in the RepB–origin complex has been investigated and it has been found that the origin undergoes significant bending movements upon RepB binding. RepB binding not only led to local bending effects but also caused a long range polar curvature, which affected the DNA sequences 3' to the H site. These movements appear to be essential for the in phase alignment of the L and H sites leading to the formation of a looped structure. A novel property of RepB unearthed in this study, is its ability to form multimers. This property may be an important factor that determines the overall trajectory of the DNA in the RepB-origin complex. The results presented in this study suggest that the origin of replication of pAL5000, and related plasmids are highly flexible and that multimeric, RepB like initiator proteins, bind and induce local deformations and long range curvatures which are probably necessary for the proper functioning of the origin.