Molecular Epidemiology and Evolution Research Programme
Project title:
TRANSPOSITIONAL DNA RECOMBINATION: MECHANISM AND UTILIZATION
Project summary:
During the last few decades it has become clear that genomes are not rigid and stable. Segments of DNA are moved, inverted, duplicated and extensively multiplied within living cells. Collectively, these phenomena are called DNA recombination. DNA transposition is a recombination reaction in which a DNA element (a transposon ) moves from one location to another in the genome of its host organism. The underlying molecular mechanisms are universal from lower prokaryotes to higher eukaryotes including retrotransposons and retroviruses. In this process specific protein molecules utilizing tightly controlled biochemical reactions excise a piece of DNA and splice it into another location in the genome. We are studying the underlying molecular mechanisms of DNA transposition using transposing bacteriophage Mu as a tool.
Bacteriophage Mu is a virus that replicates by using the mechanisms of DNA transposition. Its transposition reactions proceed within the context of specific protein DNA complexes, in the heart of which is the critical functional and structural component, the transposase. In Mu this protein is MuA and it catalyzes the DNA cleavage and joining reactions. It functions in transposition as a tetramer which synapses the two transposon ends (1,2).
Mu transposition can be studied in vitro using purified components. Lately, we have shown that under certain reaction conditions fully functional Mu transposition complexes (transpososomes ) can be formed by using short synthetic Mu-specific DNA segments and MuA protein (2). When the active transposition complex has been formed it can react, in principle, with any other DNA molecule (target DNA). We have also elucidated the role of the catalytic monomers within the transpososome regarding the two chemical steps of transposition: donor DNA cleavage and strand transfer. The results explain why formation of the transpososome is such an essential step in transposition (3).
Because we now can make DNA transposition complexes from minimal components in vitro this offers an excellent opportunity to study the governing principles in transposition at the molecular level. We will continue studying the function of transpososomes and next focus on the process of target DNA recognition. We are also studying the use of DNA transposition complexes a novel tools in molecular biology /gene technology. The in vitro transposition technology offers new ways of studying genes and their respective proteins in any experimental system.
The mechanism of DNA transposition in eukaryotic organisms is less well characterized compared to that in bacterial world; on the other hand, similarities between them are apparent and the in vitro tools to study the phenomena are very similar in both cases. In collaboration with the group of Dr . Alan Schulman, we have, therefore, initiated the elucidation of molecular mechanism of retrotransposition in plants, particularly that of the BARE -1 element of barley.
Project coordinator:
Harri Savilahti, Ph.D., Docent in Molecular Genetics, Academy of Finland Junior Fellow, Group leader, Institute of Biotechnology
Personnel:
Aalto Juha-Matti, student, Haapa Saija, M.Sc., Heikkinen Eini, M.Sc., Lamberg Arja, Ph.D., Lyra Anna, student, Mattinen Petteri, Nieminen Sari, technician, Mykkänen Anna-Helena, M.Sc., Taira Suvi, Ph.D. docent, Vilen Heikki, student
Selected publications:
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