Ziel
Two properties of the N15 prophage combine to make it unique - it inhabits a well-characterized and manageable bacterium (E.coli) and it is stably maintained as a linear plasmid with covalently closed hairpin ends (or telomeres), more akin to eukaryotic replicons than the classic circular ones of bacteria. This structure suggests that the replication of N15 requires a mechanism rarely encountered among stable bacterial replicons, while allowing access E.coli molecular genetics to facilitate its study, a convenience not afforded by the other bacterium (Borrelia spp) in which hairpin-ended linear replicons have been found. In addition, N15 is a prime example of the genetic mosaicism believed to result from horizontal transfer during evolution, and it would serve as an excellent starting point for tracing the events, which led to its present-day structure. Its maintenance functions in particular bear the hallmarks of horizontal transfer. First, the N15 replication region is very similar to that of the P4 satellite virus, which can adopt a plasmid existence, and the transcriptional control region, which regulates expression of the replication protein gene, repA, also has some parallels with that of P4. Second, the segregational stability (partition) of N15 is assured by functions very similar to the sop partition system of the F plasmid.
Replication and partition are regulated independently in the vast majority of plasmids studied, but in N15 these processes appear to interact.
The programme envisaged brings together four groups: two Russian groups who are largely responsible for our present knowledge of N15 biology, and two groups whose interests are centred on the replication of P4 (Italy) and the partition of the F plasmid (France). This evident complementarity will be focused in an effort to deepen understanding of linear replicon maintenance and the relationship between replication, telomere formation and partition.
The programme will consist of two main tasks. The first will be concentrated on the control of replication and the mechanism of telomere formation. Control factors to be investigated include the major N15 transcriptional repressor, antisense RNAs, protein modulators of RepA activity, such as that found in P4, and the sop system, one of whose centromere sites is present in the replication gene. The P4 replication protein modulation system will itself be studied in anticipation of its relevance to N15. The RepA protein is large and contains motifs characteristic of helicases, primases and DNA-binding proteins; the expression of these activities by RepA will be investigated. There are indications that replication functions other than those known exist; these will be characterized with respect to both plasmid and lytic replication. The enzyme responsible for telomere formation will also be studied.
Location of functional domains in protelomerase for telomere binding, cleavage and joining, as well as for dimerization, will be determined by site-specific mutagenesis and two-hybrid analysis. The abatement of telomerase activity during lytic replication will be studied by analysis of replication intermediates.
The second task will be focused on partition functions, and particularly on the role(s) of centromere dispersal which characterizes the N15 sop system. Dispersal may be needed to condense the linear DNA to allow efficient movement through the cytoplasm. It may also allow the centromere sequences to serve other purposes, especially transcriptional regulation. Both aspects will be studied. The results should reveal mechanistic links between these maintenance processes, aid understanding of molecular adaptation in evolution, and provide insights into the origin of linear DNA in bacteria.
More generally, the results could aid the search for new targets in combatting pathogens such as B.burgdorferi the causative agent of Lyme disease, many of whose multiple replicons are hairpin-ended linear DNAs like that of N15.
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31062 Toulouse
Frankreich