Molecular Adaptation/Evolution of an Initiation Apparatus of DNA Replication

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15570005
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Genetics/Genome dynamics
• Research Institution: University of Fukui (2004); 福井医科大学 (2003)
• Principal Investigator: INUZUKA Manabu University of Fukui, Medicine, Professor, 医学部, 教授 (00135104)
• Co-Investigator(Kenkyū-buntansha): FUJII Yutaka University of Fukui, Medicine, Associate Professor, 医学部, 助教授 (80211522)
• Project Period (FY): 2003 – 2004
• Keywords: Initiation of replication / Molecular evolution / Plasmid / Replicon / DnaA protein / Insertion sequence IS5 / Initiator protein / Reservoir model

Research Abstract

An initiation event of DNA replication is formation of a complex (initiosome) consisting of a replication origin(ori) DNA and many replication proteins. Using drug resistance plasmid R6K as a model system, we have investigated how R6K replicon adopts to the changing environments like deficiency in a DnaA protein or histon-like IHF protein that are essential for the initiation of replication. The initiation also requires two replicon-specific determinants : the ori DNA and R6K-encoded initiator π protein.
We here isolated and characterized DnaA-or IHF-independent replication mutants of R6K.
(A)Molecular adaptation to DnaA-independent initiation activity in R6K replicon due to transposition of IS5 (insertion sequence) : IS5s were transposed onto upstream regions of oriγ, from host E.coli chromosome. Both ins5A promoter region and "bending locus" of IS5 were involved in this specific function. Together with mutation analysis of the promoter activity, our results suggest that the transcripti onal activation of the initiation event by the transposed IS5 is induced at oriγ region for R6K DNA replication regulated by π protein even in the absence of DnaA protein.
(B)Specific mutations in a protein overcomes dependence on IHF protein on initiation of R6K DNA replication. IHF-independent replication mutants were all mapped on the π protein gene. These mutated π proteins showed higher binding affinity to iteron DNA present in the ori. Our data suggests that the mutated π proteins gave similar topology of the ori region even in the absence of IHF protein to that formed by the wild type π and IHF proteins.
Thus, it is the most exciting that the two types of mutations are just corresponding to two replicon-specific determinants. Furthermore, "Evolution/adaptation in a Plasmid and Bacteria Association" is clearly indicated in the initiation apparatus of DNA replication for R6K replicon. In addition to finding of the new function of IS5, it is shown that the R6K replicon always prepares the reservoir of the spontaneous mutants by changing its own genome structure, to respond to the alteration of the initiation apparatus caused by the host cells.