Deliverable D1.2

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Mol Switch Project Aim Project Description Participant List Innovation of Project Annual Reports EU policies WorkPlan 1st WorkShop Project Outcomes Workpackage No. 1 _ Deliverable 1.1 _ Deliverable 1.2 News Releases

Mol Switch is supported by funding from the IST Programme of the European Union
Mol Switch is part of the Future and Emerging Technologies Scheme

The Mol Switch Project Outcomes

Deliverable 1.2: Mutagenesis of the motor subunit HsdR(EcoprrI)

Figure 4
Conserved region in the HsdR polypeptide
of restriction enzymes EcoKI, EcoAI and EcoR124I.

As previously described for Deliverable D1.1, site-directed mutagenesis will also be used to mutate the hsdR gene from the closely related EcoprrI.

Outcomes:

All cleavage mutants of HsdR(EcoprrI) subunit were purified and mutant endonucleases reconstituted from the wt methylase and individual mutant HsdR subunits were analysed in vitro. Cleavage assay proved in agreement with the in vivo phenotype that none of the mutant enzyme is able to cleave the plasmid DNA to its linear form. DNA binding, ATPase and translocation activities are similar to the wt enzyme.

References:

Janscak, P., Sandmeier, U., and Bickle, T.A. (1999) Single amino acid substitutions in the HsdR subunit of the type IB restriction enzyme EcoAI uncouple the DNA translocation and DNA cleavage activities of the enzyme. N.A.R. 27: 2638-2643.

Davies, G.P., Kemp, P., Molineux, I.J., and Murray, N.E. (1999) The DNA translocation and ATPase activities of restriction-deficient mutants of EcoKI. JMB 292: 787-796.

Ohmori, H. (1994) A new method for strand discrimination in sequence-directed mutagenesis. N.A.R. 22: 884-885.

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