Deliverable 1.3:
Assembly mutation studies with hsdR
Full Report
PCR-based mis-incorporation mutagenesis (Arcangioli et al.,
1994) will be used to study the effect of single and multiple
point mutations within the N-terminal region of the motor
protein. This region is important for correct assembly on to
the MTase of the final HsdR subunit, producing the R1-complex,
therefore mutations in this region are likely to produce a
more stable R1-complex. This would provide us with a
unidirectional motor and together with the above mutations in
motif-X ensure the motor cannot cut DNA. Therefore, this work
will provide a better understanding of the assembly process
with an aim of producing assembly in a defined orientation
with regard to the asymmetric recognition sequence. Such a
system would provide a unidirectional motor with a
predetermined direction built in.
Outcomes:
Eleven mutant hsdR genes have been prepared. Substitutions
E141A, T280P, D372A, D375A and D374A were confirmed by DNA
sequencing and provided for in vitro analysis.
Sequence analysis of other six substitutions D341A, E610A, E618A,
D613A, D630 and D640A revealed a secondary mutation in the hsdR
gene. With regards to this fact and to general difficulties with
the plasmid pGEX-2T, when even plasmid with wt hsdR gene
expresses very low restriction activity in vivo (10-1),
further mutagenic work with this plasmid was stopped.
References:
Arcangioli, B., Ghazvini, M., and Ribes, V. (1994) Identification of
the DNA-binding domains of the switch-activating-protein Sap1 from S.
pombe by random point mutations screening in E. coli. N.A.R.22: 2930-2937.
Project Outcomes
