Biochemical Investigations of Control of Replication Initiation of Plasmid R6K

Mayuresh M Abhyankar,Jagan M Reddy,Rahul Sharma,Erika Büllesbach,Deepak Bastia

doi:10.1074/jbc.m312052200

Mayuresh M Abhyankar, Jagan M Reddy + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m312052200

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Abstract

The mechanistic basis of control of replication initiation of plasmid R6K was investigated by addressing the following questions. What are the biochemical attributes of mutations in the pi initiator protein that caused loss of negative control of initiation? Did the primary control involve only initiator protein-ori DNA interaction or did it also involve protein-protein interactions between pi and several host-encoded proteins? Mutations at two different regions of the pi-encoding sequence individually caused some loss of negative control as indicated by a relatively modest increase in copy number. However, combinations of the mutation P42L, which caused loss of DNA looping, with those located in the region between the residues 106 and 113 induced a robust enhancement of copy number. These mutant forms promoted higher levels of replication in vitro in a reconstituted system consisting of 22 purified proteins. The mutant forms of pi were susceptible to pronounced iteron-induced monomerization in comparison with the WT protein. As contrasted with the changes in DNA-protein interaction, we found no detectable differences in protein-protein interaction between wild type pi with DnaA, DnaB helicase, and DnaG primase on one hand and between the high copy mutant forms and the same host proteins on the other. The DnaG-pi interaction reported here is novel. Taken together, the results suggest that both loss of negative control due to iteron-induced monomerization of the initiator and enhanced iteron-initiator interaction appear to be the principal causes of enhanced copy number.

Highlights

Ever since the publication of the landmark “Replicon Model” 40 years ago by Jacob, Brenner, and Cuzin [1], molecular analyses of the regulation of copy control of plasmid DNAs have been and continue to be instrumental in providing key insights into the mechanisms of regulation of replication initiation
As contrasted with the changes in DNAprotein interaction, we found no detectable differences in protein-protein interaction between wild type ␲ with DnaA, DnaB helicase, and DnaG primase on one hand and between the high copy mutant forms and the same host proteins on the other
Our observations show that (i) the high copy mutations reduced negative control by reducing or eliminating handcuffing and by enhancing the interaction of the protein with ori DNA; (ii) the enhanced binding was probably caused by DNA-ligand-induced monomerization of the high copy mutant forms of ␲ as contrasted with the wild type (WT) protein that did not monomerize upon DNA binding; and (iii) there was no measurable differences in binary interactions between the wt ␲ with host-encoded DnaA, DnaB helicase, and DnaG primase on one hand and between the mutant forms with the same host proteins on the other

Summary

Introduction

Ever since the publication of the landmark “Replicon Model” 40 years ago by Jacob, Brenner, and Cuzin [1], molecular analyses of the regulation of copy control of plasmid DNAs have been and continue to be instrumental in providing key insights into the mechanisms of regulation of replication initiation. A second, more ubiquitous mechanism of copy control is by interaction of the plasmid encoded initiator protein with short tan-. Demly repeated sequences called iterons, which are present at the replication origins of bacterial and plasmid chromosomes (6 –9). Using topoisomerase II-mediated formation of catenated dimers, we showed previously that initiator protein promotes pairing of two replication origins in vitro [21]. Helinski and independently Chattoraj were the first to point out that the initiator protein-mediated pairing of plasmid DNA at the iterons in vivo either in cis or in trans causes turning off of replication initiation, and this mechanism of repression is called “handcuffing” [17, 18]. We postulate that activation of ␤ (or ␣) requires a single dimeric ␲ and monomeric ␲ proteins that are bound to the remainder of the ␥ iterons and are involved in the formation of a preinitiation complex that includes DnaA (see Fig.1B)

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